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Bacillus thuringiensis is an insect pathogen that is widely used as a biopesticide (3). Here we report the finished, annotated genome sequence of B. thuringiensis Al Hakam, which was collected in Iraq by the United Nations Special Commission (2).

BackgroundClostridium botulinum and related clostridial species express extremely potent neurotoxins known as botulinum neurotoxins (BoNTs) that cause long-lasting, potentially fatal intoxications in humans and other mammals. The amino acid variation within the BoNT is used to categorize the species into seven immunologically distinct BoNT serotypes (A-G) which are further divided into subtypes. The BoNTs are located within two generally conserved gene arrangements known as botulinum progenitor complexes which encode toxin-associated proteins involved in toxin stability and expression.Methodology/principal findingsBecause serotype A and B strains are responsible for the vast majority of human botulism cases worldwide, the location, arrangement and sequences of genes from eight different toxin complexes representing four different BoNT/A subtypes (BoNT/A1-Ba4) and one BoNT/B1 strain were examined. The bivalent Ba4 strain contained both the BoNT/A4 and BoNT/bvB toxin clusters. The arrangements of the BoNT/A3 and BoNT/A4 subtypes differed from the BoNT/A1 strains and were similar to those of BoNT/A2. However, unlike the BoNT/A2 subtype, the toxin complex genes of BoNT/A3 and BoNT/A4 were found within large plasmids and not within the chromosome. In the Ba4 strain, both BoNT toxin clusters (A4 and bivalent B) were located within the same 270 kb plasmid, separated by 97 kb. Complete genomic sequencing of the BoNT/B1 strain also revealed that its toxin complex genes were located within a 149 kb plasmid and the BoNT/A3 complex is within a 267 kb plasmid.Conclusions/significanceDespite their size differences and the BoNT genes they contain, the three plasmids containing these toxin cluster genes share significant sequence identity. The presence of partial insertion sequence (IS) elements, evidence of recombination/gene duplication events, and the discovery of the BoNT/A3, BoNT/Ba4 and BoNT/B1 toxin complex genes within plasmids illustrate the different mechanisms by which these genes move among diverse genetic backgrounds of C. botulinum.

The role of Real-Time PCR assays for surveillance and rapid screening for pathogens is garnering more and more attention because of its versatility and ease of adoption. The goal of this study was to design, test, and evaluate Real-Time TaqMan PCR assays for the detection of botulinum neurotoxin (bont/A-G) genes from currently recognized BoNT subtypes. Assays were computationally designed and then laboratory tested for sensitivity and specificity using DNA preparations containing bont genes from 82 target toxin subtypes, including nine bivalent toxin types; 31 strains representing other clostridial species; and an extensive panel that consisted of DNA from a diverse set of prokaryotic (bacterial) and eukaryotic (fungal, protozoan, plant, and animal) species. In addition to laboratory testing, the assays were computationally evaluated using in silico analysis for their ability to detect bont gene sequences from recently identified toxin subtypes. Seventeen specific assays (two for each of the bont/C, bont/D, bont/E, and bont/G subtypes and three for each of the bont/A, bont/B, and bont/F subtypes) were designed and evaluated for their ability to detect bont genes encoding multiple subtypes from all seven serotypes. These assays could provide an additional tool for the detection of botulinum neurotoxins in clinical, environmental and food samples that can complement other existing methods used in clinical diagnostics, regulatory, public health, and research laboratories. [ABSTRACT FROM AUTHOR]

The sequencing and analysis of two close relatives of Bacillus anthracis are reported. AFLP analysis of over 300 isolates of B. cereus, B. thuringiensis and B. anthracis identified two isolates as being very closely related to B. anthracis. One, a B. cereus, BcE33L, was isolated from a zebra carcass in Nambia; the second, a B. thuringiensis, 97-27, was isolated from a necrotic human wound. The B. cereus appears to be the closest anthracis relative sequenced to date. A core genome of over 3,900 genes was compiled for the Bacillus cereus group, including B anthracis. Comparative analysis of these two genomes with other members of the B. cereus group provides insight into the evolutionary relationships among these organisms. Evidence is presented that differential regulation modulates virulence, rather than simple acquisition of virulence factors. These genome sequences provide insight into the molecular mechanisms contributing to the host range and virulence of this group of organisms.

Sequencing of the genome of Clostridium botulinum strain Hall A revealed a gene (CBO0515), whose putative amino acid sequence was suggestive of the rare enzyme [N.sup.5]-(1-carboxyethyl) ornithine synthase. To test this hypothesis, CBO0515 has been cloned, and the encoded polypeptide was purified and characterized. This unusual gene appears to be confined to proteolytic strains assigned to group 1 of C. botulinum. doi:10.1128/JB.01044-09

A small number of conserved canonical single nucleotide polymorphisms (canSNP) that define major phylogenetic branches for Bacillus anthracis were used to place a Sverdlovsk patient's B. anthracis genotype into 1 [...]

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are closely related gram-positive, spore-forming bacteria of the B. cereus sensu lato group. While independently derived strains of B. anthracis reveal conspicuous sequence homogeneity, environmental isolates of B. cereus and B. thuringiensis exhibit extensive genetic diversity. Here we report the sequencing and comparative analysis of the genomes of two members of the B. cereus group, B. thuringiensis 97-27 subsp, konkukian serotype H34, isolated from a necrotic human wound, and B. cereus E33L, which was isolated from a swab of a zebra carcass in Namibia. These two strains, when analyzed by amplified fragment length polymorphism within a collection of over 300 of B. cereus, B. thuringiensis, and B. anthracis isolates, appear closely related to B. anthracis. The B. cereus E33L isolate appears to be the nearest relative to B. anthracis identified thus far. Whole-genome sequencing of B. thuringiensis 97-27 and B. cereus E33L was undertaken to identify shared and unique genes among these isolates in comparison to the genomes of pathogenic strains B. anthracis Ames and B. cereus G9241 and nonpathogenic strains B. cereus ATCC 10987 and B. cereus ATCC 14579. Comparison of these genomes revealed differences in terms of virulence, metabolic competence, structural components, and regulatory mechanisms.

Bacillus thuringiensis, Bacillus cereus and Bacillus anthracis are three closely related species of the B.cereus group with each of them being economically important and their DNA isolates were analyzed using fluorescent amplified fragment length polymorphism (AFLP). Phytogenetic analysis of AFLP data showed diversity with B.thuringinesis and B.cereus compared to the monomorphic nature of B.anthracis.

Researchers used fluorescent amplified fragment length polymorphism to analyze 154 Bacillus cereus and Bacillus thuringiensis isolates taken from five different locations and compare them to reference strains. This method can be automated, allowing scientists to process very large samples.

The construction of a small-scale, non-selective procedure for DNA sample preparation supporting rapid-in-the-field polymerase chain reaction analysis for detection of microbial spores and cells is described. Three methods capable of releasing genomic DNA from three microorganisms were chosen for cell and spore disruption techniques. The reagents and buffers used in the three methods were changed and the techniques are miniaturized to provide maximum DNA yields.

A bacterial biosensor for benzene, toluene and similar compounds appears to be capable of accurately measuring environmental contaminants, making it a potentially inexpensive tool for field-ready assays. A reporter plasmid was constructed by placing the luc gene for firefyl luciferase directly under the control of the P(sub u) promoter and the xylR transcriptional activator of Pseudomonas putida mt-2. The biosensor cells were shown to detect benzene, toluene and xylene contamination in soil and water samples with high sensitivity and quantitative accuracy.

PCR analysis of 198 Bacillus anthracis isolates indicated a variable region of DNA sequence differing in length among the isolates. Five polymorphisms were characterized by the presence of two to six copies of the 12-bp tandem repeat 5'-CAATATCAACAA-3'. This variable-number tandem repeat region is found within a larger sequence containing one complete open reading frame that encodes a putative 30-kDa protein. Length variation did not alter the reading frame of the encoded protein and only modified the copy number of a 4-amino-acid sequence (QYQQ) from 2 to 6.

Abstract Background An isolate originally labeled Bacillus megaterium CDC 684 was found to contain both pXO1 and pXO2, was non-hemolytic, sensitive to gamma-phage, and produced both the protective antigen and the poly-D-glutamic acid capsule. These phenotypes prompted Ezzell et al., (J. Clin. Microbiol. 28:223) to reclassify this isolate to Bacillus anthracis in 1990. Results We demonstrate that despite these B. anthracis features, the isolate is severely attenuated in a guinea pig model. This prompted whole genome sequencing and closure. The comparative analysis of CDC 684 to other sequenced B. anthracis isolates and further analysis reveals: a) CDC 684 is a close relative of a virulent strain, Vollum A0488; b) CDC 684 defines a new B. anthracis lineage (at least 51 SNPs) that includes 15 other isolates; c) the genome of CDC 684 contains a large chromosomal inversion that spans 3.3 Mbp; d) this inversion has caused a displacement of the usual spatial orientation of the origin of replication (ori) to the termination of replication (ter) from 180° in wild-type B. anthracis to 120° in CDC 684 and e) this isolate also has altered growth kinetics in liquid media. Conclusions We propose two alternative hypotheses explaining the attenuated phenotype of this isolate. Hypothesis 1 suggests that the skewed ori/ter relationship in CDC 684 has altered its DNA replication and/or transcriptome processes resulting in altered growth kinetics and virulence capacity. Hypothesis 2 suggests that one or more of the single nucleotide polymorphisms in CDC 684 has altered the expression of a regulatory element or other genes necessary for virulence.

Abstract Background Clostridium botulinum is a taxonomic designation for at least four diverse species that are defined by the expression of one (monovalent) or two (bivalent) of seven different C. botulinum neurotoxins (BoNTs, A-G). The four species have been classified as C. botulinum Groups I-IV. The presence of bont genes in strains representing the different Groups is probably the result of horizontal transfer of the toxin operons between the species. Results Chromosome and plasmid sequences of several C. botulinum strains representing A, B, E and F serotypes and a C. butyricum type E strain were compared to examine their genomic organization, or synteny, and the location of the botulinum toxin complex genes. These comparisons identified synteny among proteolytic (Group I) strains or nonproteolytic (Group II) strains but not between the two Groups. The bont complex genes within the strains examined were not randomly located but found within three regions of the chromosome or in two specific sites within plasmids. A comparison of sequences from a Bf strain revealed homology to the plasmid pCLJ with similar locations for the bont/bv b genes but with the bont/a4 gene replaced by the bont/f gene. An analysis of the toxin cluster genes showed that many recombination events have occurred, including several events within the ntnh gene. One such recombination event resulted in the integration of the bont/a1 gene into the serotype toxin B ha cluster, resulting in a successful lineage commonly associated with food borne botulism outbreaks. In C. botulinum type E and C. butyricum type E strains the location of the bont/e gene cluster appears to be the result of insertion events that split a rarA, recombination-associated gene, independently at the same location in both species. Conclusion The analysis of the genomic sequences representing different strains reveals the presence of insertion sequence (IS) elements and other transposon-associated proteins such as recombinases that could facilitate the horizontal transfer of the bonts; these events, in addition to recombination among the toxin complex genes, have led to the lineages observed today within the neurotoxin-producing clostridia.

Variable-number tandem-repeat (VNTR) markers are used for differentiating among the clostridial strains. These markers have provided a rapid, highly discriminatory tool for distinguishing among Clostridium botulinum botulism neurotoxin A1 (BoNT/A1) strains for examining botulism outbreaks.

Research has been conducted on genetically related group of Bacillus anthracis, B. cereus and B. thuringiensis. The authors discuss the phylogenetic relationships between these non-anthrax-causing Bacillus species.

Bacillus thuringiensis is an insect pathogen that is widely used as a biopesticide (E. Schnepf, N. Crickmore, J. Van Rie, D. Lereclus, J. Baum, J. Feitelson, D. R. Zeigler, and D. H. Dean, Microbiol. Mol. Biol. Rev. 62:775-806, 1998). Here we report the finished, annotated genome sequence of B. thuringiensis Al Hakam, which was collected in Iraq by the United Nations Special Commission (L. Radnedge, P. Agron, K. Hill, P. Jackson, L. Ticknor, P. Keim, and G. Andersen, Appl. Environ. Microbiol. 69:2755-2764, 2003). doi:10.1128/JB.00241-07

Botulinum neurotoxin (BoNT) is the most poisonous substances known and its eight toxin types (A to H) are distinguished by the inability of polyclonal antibodies that neutralize one toxin type to neutralize any of the other seven toxin types. Infant botulism, an intestinal toxemia orphan disease, is the most common form of human botulism in the United States. It results from swallowed spores of Clostridium botulinum (or rarely, neurotoxigenic Clostridium butyricum or Clostridium baratii) that germinate and temporarily colonize the lumen of the large intestine, where, as vegetative cells, they produce botulinum toxin. Botulinum neurotoxin is encoded by the bont gene that is part of a toxin gene cluster that includes several accessory genes. We sequenced for the first time the complete botulinum neurotoxin gene cluster of nonproteolytic C. baratii type F7. Like the type E and the nonproteolytic type F6 botulinum toxin gene clusters, the C. baratii type F7 had an orfX toxin gene cluster that lacked the regulatory botR gene which is found in proteolytic C. botulinum strains and codes for an alternative σ factor. In the absence of botR, we identified a putative alternative regulatory gene located upstream of the C. baratii type F7 toxin gene cluster. This putative regulatory gene codes for a predicted σ factor that contains DNA-binding-domain homologues to the DNA-binding domains both of BotR and of other members of the TcdR-related group 5 of the σ70 family that are involved in the regulation of toxin gene expression in clostridia. We showed that this TcdR-related protein in association with RNA polymerase core enzyme specifically binds to the C. baratii type F7 botulinum toxin gene cluster promoters. This TcdR-related protein may therefore be involved in regulating the expression of the genes of the botulinum toxin gene cluster in neurotoxigenic C. baratii. [ABSTRACT FROM AUTHOR]

Sanger and shotgun sequencing of Clostridium botulinum strain Af84 type Af and its botulinum neurotoxin gene (bont) clusters identified the presence of three bont gene clusters rather than the expected two. The three toxin gene clusters consisted of bont subtypes A2, F4 and F5. The bont/A2 and bont/F4 gene clusters were located within the chromosome (the latter in a novel location), while the bont/F5 toxin gene cluster was located within a large 246 kb plasmid. These findings are the first identification of a C. botulinum strain that contains three botulinum neurotoxin gene clusters. [ABSTRACT FROM AUTHOR]

In May of 2011, an enteroaggregative Escherichia coli O104:H4 strain that had acquired a Shiga toxin 2-converting phage caused a large outbreak of bloody diarrhea in Europe which was notable for its high prevalence of hemolytic uremic syndrome cases. Several studies have described the genomic inventory and phylogenies of strains associated with the outbreak and a collection of historical E. coli O104:H4 isolates using draft genome assemblies. We present the complete, closed genome sequences of an isolate from the 2011 outbreak (2011C-3493) and two isolates from cases of bloody diarrhea that occurred in the Republic of Georgia in 2009 (2009EL-2050 and 2009EL-2071). Comparative genome analysis indicates that, while the Georgian strains are the nearest neighbors to the 2011 outbreak isolates sequenced to date, structural and nucleotide-level differences are evident in the Stx2 phage genomes, the mer/tet antibiotic resistance island, and in the prophage and plasmid profiles of the strains, including a previously undescribed plasmid with homology to the pMT virulence plasmid of Yersinia pestis. In addition, multiphenotype analysis showed that 2009EL-2071 possessed higher resistance to polymyxin and membrane-disrupting agents. Finally, we show evidence by electron microscopy of the presence of a common phage morphotype among the European and Georgian strains and a second phage morphotype among the Georgian strains. The presence of at least two stx2 phage genotypes in host genetic backgrounds that may derive from a recent common ancestor of the 2011 outbreak isolates indicates that the emergence of stx2 phage-containing E. coli O104:H4 strains probably occurred more than once, or that the current outbreak isolates may be the result of a recent transfer of a new stx2 phage element into a pre-existing stx2-positive genetic background. [ABSTRACT FROM AUTHOR]

Sequencing of the genome of Clostridium botulinum strain Hall A revealed a gene (CBO0515), whose putative amino acid sequence was suggestive of the rare enzyme N5-(1-carboxyethyl) ornithine synthase. To test this hypothesis, CBO0515 has been cloned, and the encoded polypeptide was purified and characterized. This unusual gene appears to be confined to proteolytic strains assigned to group 1 of C. botulinum. [ABSTRACT FROM AUTHOR]