Identification of a Bacillus anthracis specific indel in the yeaC gene and development of a rapid pyrosequencing assay for distinguishing B. anthracis from the B. cereus group

Journal of Microbiological Methods 87 (2011) 278–285

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Identification of a Bacillus anthracis specific indel in the yeaC gene and development of a rapid pyrosequencing assay for distinguishing B. anthracis from the B. cereus group Nadia Z. Ahmod a,⁎, Radhey S. Gupta b, Haroun N. Shah a a b

Department for Bioanalysis and Horizon Technologies, Centre for Infections, Health Protection Agency, 61 Colindale Avenue, Colindale, London, NW9 5EQ United Kingdom Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Canada L8N 3Z5

a r t i c l e

i n f o

Article history: Received 17 May 2011 Received in revised form 22 August 2011 Accepted 22 August 2011 Available online 2 September 2011 Keywords: B. anthracis B. cereus YeaC protein Anthracis-specific indel Pyrosequencing Rapid detection

a b s t r a c t Bacillus anthracis, the causative agent of anthrax, is a potential source of bioterrorism. The existing assays for its identification lack specificity due to the close genetic relationship it exhibits to other members of the B. cereus group. Our comparative analyses of protein sequences from Bacillus species have identified a 24 amino acid deletion in a conserved region of the YeaC protein that is uniquely present in B. anthracis. PCR primers based on conserved regions flanking this indel in the Bacillus cereus group of species (viz. Bacillus cereus, B. anthracis, B. thuringiensis, B. mycoides, B. weihenstephnensis and B. pseudomycoides) specifically amplified a 282 bp fragment from all six reference B. anthracis strains, whereas a 354 bp fragment was amplified from 15 other B. cereus group of species/strains. These fragments, due to large size difference, are readily distinguished by means of agarose gel electrophoresis. In contrast to the B. cereus group, no PCR amplification was observed with any of the non-B. cereus group of species/strains. This indel was also used for developing a rapid pyrosequencing assay for the identification of B. anthracis. Its performance was evaluated by examining the presence or absence of this indel in a panel of 81 B. cereus-like isolates from various sources that included 39 B. anthracis strains. Based upon the sequence data from the pyrograms, the yeaC indel was found to be a distinctive characteristic of various B. anthracis strains tested and not found in any other species/strains from these samples. Therefore, this B. anthracis specific indel provides a robust and highly-specific chromosomal marker for the identification of this high-risk pathogen from other members of the B. cereus group independent of a strain's virulence. The pyrosequencing platform also allows for the rapid and simultaneous screening of multiple samples for the presence of this B. anthracis-specific marker. © 2011 Published by Elsevier B.V.

1. Introduction Bacillus anthracis is the causative agent of anthrax, which is a lethal disease of animals and humans (Mock and Fouet, 2001; Kolstø et al., 2009). The genus Bacillus comprises species that are Gram positive, rod shaped, aerobic or facultatively anaerobic, endospore-forming organisms that are catalase positive (Fritze, 2004; Drobniewski, 1993; Blackwood et al., 2004). B. anthracis is a part of the B. cereus group, which is a subgroup of Bacillus species that are very closely related by 16S rRNA gene analysis (Ash et al., 1991). This group consists of six recognised species, B. cereus, B. anthracis, B. thuringiensis, B. mycoides, B. pseudomycoides and B. weihenstephanensis (Bavykin et al., 2004; Lechner et al., 1998; Nakamura and Jackson, 1995; Jensen et al., 2003). Of these, B. pseudomycoides, B. mycoides and B. weihenstephanensis are regarded as non-pathogenic whereas B. anthracis, B. cereus and B. thuringiensis are opportunistic or pathogenic to insects or mammals (Barker et al., 2005; Kolstø et al., 2009). B. cereus is a common food-borne ⁎ Corresponding author. Tel.: + 44 208 327 7229; fax: + 44 208 327 7870. E-mail addresses: [email protected] (N.Z. Ahmod), [email protected] (R.S. Gupta), [email protected] (H.N. Shah). 0167-7012/$ – see front matter © 2011 Published by Elsevier B.V. doi:10.1016/j.mimet.2011.08.015

pathogen that causes diarrhoea and vomiting and produces several toxins encoded on the chromosome and plasmids that contribute to its virulence (Barker et al., 2005). However, some of the B. cereus virulence factors such as genes encoding enterotoxins may also be found in strains of B. anthracis and B. thuringiensis (Han et al., 2006; Hansen and Hendriksen, 2001; Read et al., 2003). B. thuringiensis is primarily an insect pathogen and is sometimes used as a biopesticide. It is distinguished from other members of the group by its ability to produce plasmid mediated crystalline, proteinaceous, parasporal bodies during sporulation (Chen and Tsen, 2002; Schnepf et al., 1998; Jensen et al., 2003). B. anthracis, which is the most deadly of these pathogens, contains two plasmids that are responsible for its virulence (Jensen et al., 2003; Kolstø et al., 2009); pXO1 encodes for three lethal toxins and pXO2 encodes for a capsule that is made up of poly-D-glutamic acid and is responsible for protecting the organism from phagocytosis (Jensen et al., 2003). Both of these plasmids are needed to cause infection in humans and if either of them is lost, the organism is no longer virulent (Jensen et al., 2003; Qi et al., 2001). B. cereus, B. anthracis and B. thuringiensis share a great deal of morphological and biochemical properties and share such a high degree of genetic similarity that several studies have proposed that they

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should all be considered as sub-species of B. cereus (Ash et al., 1991; Helgason et al., 2000; Priest et al., 2004). It has been further suggested that their pathogenic properties and virulence factors may be used for distinguishing them at the species level (La Duc et al., 2004). However, due to the high degree of lateral gene transfer of these virulence factors and the fact that some of these genes are encoded on plasmids that can be lost by the organism, difficulties exist when attempting to accurately determine their identity to the species level. Because of their varying pathogenic properties and the increasing awareness of their pathogenic potential (particularly of B. anthracis), it is essential to accurately delineate these taxa. Molecular based methods such as analysis of the 16S rRNA, gyrA and gyrB gene, pulse field gelelectrophoresis analysis and multilocus sequence typing (MLST) that target the chromosome have failed to provide consistent differences between isolates to enable their identification (Ash et al., 1991; Bavykin et al., 2004; Carlson et al., 1994; Hurtle et al., 2004; Priest et al., 2004). For the identification of B. anthracis, two virulence plasmids have been targeted. PCR based assays that target these plasmids are able to distinguish fully virulent B. anthracis containing both plasmids from those that contain either pX01 or pX02. However, based on these gene targets, the plasmid cured strains of B. anthracis cannot be identified; additionally, closely related strains of B. cereus and B. thuringiensis that contain genes derived from the B. anthracis plasmids can yield false positive results (Ellerbrok et al., 2002). In view of these problems, chromosomal markers, that offer greater specificity for B. anthracis should enable clear demarcation of this species from closely related members of the B. cereus group including species that may harbour some of the common plasmids. Although a number of chromosomal markers based on rpoB, gyrA, gyrB, plcR,

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saspB, BA5345 and BA813, have been reported to distinguish B. anthracis (Ko et al., 2003; Oggioni et al., 2002; Qi et al., 2001; Hurtle et al., 2004; Ramisse et al., 1999), concerns have been raised about their ability to distinguish B. anthracis from closely related B. cereus strains (Rao et al., 2010). It is therefore important to identify other chromosomal markers that offer greater specificity in distinguishing this important human pathogen from other closely related species/strains. The availability of genome sequences from diverse groups of microorganisms provides an unprecedented resource for discovery of molecular markers that are specific for different groups of organisms and provide novel tools for evolutionary, genetic and diagnostic purposes. The conserved indels (i.e. insert or deletion) in protein sequences provide one important class of molecular markers that have proven very useful for such studies (Gupta and Griffiths, 2002, 2006). Recent work in this area has identified large numbers of conserved indels that are specific for different main groups/taxa of bacteria (e.g. Chlamydiae, Alphaproteobacteria, Gammaproteobacteria, Cyanobacteria, Bacteroidetes, Aquificae, Actinobacteria, Clostridium, etc.) and they provide a novel means for the identification and circumscription of these groups of bacteria in more definitive molecular terms (Griffiths et al., 2005; Gupta and Lorenzini, 2007; Gupta and Mok, 2007; Gao et al., 2009; Gupta, 2009; Gupta and Gao, 2009). Although the indel approach in the past has mainly been used for identification of taxonomic clades at genus or higher levels, this approach is equally applicable for identification of individual species and strains. In the present work, we report the application of this approach to identify a large conserved indel that is specific for the B. anthracis species/strains. We present evidence that this large indel provides a highly specific marker for reliably distinguishing this

Table 1 List of Bacillus strains used in the study. Group

Species

B. cereus group

B. anthracis B. cereus B. thuringiensis B. mycoides B. weihenstephanensis B. pseudomycoides Other Bacillus spp. and closely B. licheniformis related genera B. subtilis

Food isolates

B. pumilus B. amyloliquefaciens B. vallismortis B. mojavensis B. sonorensis B. megaterium B. badius B. simplex B. firmus B. lentus B. clausii B. circulans L. sphaericus L. fusiformis P. macerans P. polymyxa V. pantothenticus Brevibacillus brevis B. cereus group

Environmental isolates

B. cereus group

Anthrax strain collection (DNA B. anthracis provided)

Strain NCTC 7753, NCTC 7752, NCTC 1328, NCTC 10340T, NCTC 109, NCTC 5444 NCTC 9939, NCTC 10987, NCTC 11143, NCTC 9945, NCTC 8035, NCTC 11145 DSM 6029, DSM 2046T, DSM 350 NCTC 9680, NCTC 7586, NCTC 12974T, NCTC 2603 DSM 11821T DSM 12442T NCTC 10341T, NCTC 06346, NCTC 7467, NCTC 1024, NCTC 1097, NCTC 2120, NCTC 9932, NCTC 1025, NCTC 5399, NCTC 6816, NCTC 7589, NCTC 8233, NCTC 8720, NCTC 8721, NCTC 0962 NCTC 6431, NCTC 9933, NCTC 10073, NCTC 10452, NCTC 3610T, NCTC 7861, NCTC 10315, NCTC 10400, NCTC 3398, NCTC 6432, NCTC 8236 NCTC 2595, NCTC 7576, NCTC 10337T, NCTC 8241, NCTC 2596, NCTC 7198, NCTC 9436, NCTC 10327 DSM 1060 DSM 11031T DSM 9205T, DSM 9206 DSM 13779T, DSM 13780 NCTC 10342T, NCTC 5635, NCTC 5637, NCTC 6005 DSM 5610, NCTC 10333T DSM 1317, DSM 1321T, NCIMB 8796 DSM 359 NCTC 4824T DSM 8716T, DSM 2512 NCTC 5846, NCTC 5895, NCTC 2610T NCTC 10338T, NCTC 2609, NCTC 5896, NCTC 7585, NCTC 2608 DSM 493, DSM 2898T NCTC 7588, NCTC 6355T NCTC 7575, NCTC 4744, NCTC 4747, NCTC 10343T NCTC 8162T, NCTC 8124 NCTC 7096 HPA10304, HPA10904, HPA48604, HPA73804, HPA54804, HPA62404, HPA23704, HPA23104, HPA23205, HPA07404, HPA64204, HPA02906, HPA05804, HPA75304, HPA31604, HPA09704, HPA10205 A4s1, A4i3, A4m3, A4s2, E5m5, A4o4, A4g6, A4s3, A4i5, E2o4, A4o3, A4m4, A4g2, C3i5, A4g3, C3i1, A4g1, A4o6, C3m4, C3o43, A4g5, C3o28, A4s6, A4s8, A4s9 ASC20, ASC31, ASC45, ASC46, ASC51, ASC60, ASC63, ASC65, ASC70, ASC72, ASC85, ASC91, ASC95, ASC117, ASC120, ASC136, ASC140, ASC149, ASC152, ASC157, ASC180, ASC191, ASC200, ASC207, ASC216, ASC215, ASC270, ASC273, ASC298, ASC319, ASC330, ASC336, ASC341, ASC379, ASC389, ASC393, ASC397, ASC401, ASC412

Type strains are listed with the culture collection number followed by a superscript “T”. Culture collection strains were obtained from NCIMB: National Collection of Industrial, Marine and Food Bacteria, NCTC: National Collection of Type Cultures, DSM: Deutsche Sammlung von Mikroorganismen.

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important pathogenic species from all other closely related bacteria belonging to the B. cereus group. 2. Materials and methods 2.1. Bacterial strains and DNA Twenty-one reference strains belonging to the B. cereus group (i.e. B. cereus, B. anthracis, B. thuringiensis, B. mycoides, B. pseudomycoides and B. weihenstephanensis) were obtained from the National Collection of Type Cultures (NCTC), German Collection of Microorganisms and Cell Cultures (DSMZ) and The National Collection of Industrial, Food and Marine Bacteria (NCIMB). Seventeen strains belonging to the B. cereus group were provided by The Food Safety Microbiology Laboratory (FSML), HPA and 25 environmental B. cereus group strains were obtained from the NERC Centre for Population Biology, Imperial College. All were previously identified as members of the B. cereus group by 16S rRNA sequence analysis. 72 reference strains belonging to non-B. cereus group species were also included in this study. Strains were cultured on Columbia Blood Agar at 37 °C for 24 h under aerobic conditions and genomic DNA was extracted using either the Zymo DNA Extraction Kit (Cambridge Bioscience) or the Prepman® Ultra Sample Preparation Reagent (Applied Biosystems) according to the manufacturer's instructions. In addition to these strains, DNA extracts from 39 B. anthracis strains from the anthrax strain collection (ASC) were provided by Health Protection Agency, Porton Down. The details of all strains are listed in Table 1. 2.2. Analyses of protein sequences in Bacillus genomes The indel that is specific for B. anthracis was identified as a part of our study aimed at identifying conserved indels that are specific for either all Bacillus spp. or different subclades of the Bacillus genus. These studies were carried out in a similar manner as described in earlier work (Griffiths et al., 2005; Gao et al., 2009; Gupta, 2009). In these studies, Blastp searches were initially carried out on various proteins (or ORFs) (N100 aa long) from the genome of Bacillus subtilis subsp. subtilis str. 168 (AL009126) using default parameters (Altschul et al., 1990). For various proteins for whom high scoring homologs were detected in at least 6 Bacillus species, the sequences for representative Bacillus spp. as well as some outgroup taxa (e.g. Staphylococcus, Streptococcus, Lactobacillus, etc.) were retrieved and their multiple sequence alignments were constructed using the clustalX programme (Thompson et al., 1997). The resulting sequence alignments were visually inspected to identify any conserved indel that was restricted to either some or all Bacillus spp., but not present in any of the outgroup species. The indels that were not flanked by conserved regions were not studied further, as they do not provide useful molecular markers (Gao et al., 2009; Gupta, 2009). The species specificity of all potential indels thus identified was further evaluated by detailed Blastp searches on a short segment of the protein sequence containing the indel and its flanking conserved regions. 2.3. PCR amplification and direct sequencing For the designing of PCR primers for the yeaC gene, a sequence alignment of the yeaC DNA sequences from various B. cereus species/ strains listed in Table 2 was constructed. The primers were designed for two conserved (consensus) sequence regions that flanked the B. anthracis indel in this alignment. PCR amplification of the target gene was performed using 1 μL of the template DNA in a 50 μL reaction containing 25 μL GoTaqGrean (Promega), 10 pmol of biotinylated forward primer BSU06330_F (5′BIO-TTT ACC AGA AGC HCA GCT CG3′) (MWG eurofins) and 10 pmol of reverse primer BSU06330_R (5′-GCT AAA AAT TTA ACA TCG TCT GGA-3′) (MWG eurofins) and the total volume made up with nuclease free water (Promega). The

reaction mixture was subjected to an initial denaturation step at 96 °C for 2 min followed by 30 cycles of denaturation at 94 °C for 45 s, annealing at 50 °C for 45 s and elongation at 72 °C for 1.5 min followed by a final elongation step at 72 °C for 10 min. Electrophoresis was performed on the PCR amplicons at 100 V for 1 h on a 2% agarose gel containing Gelred (Biotium) for visualisation. A 100 bp DNA ladder (Promega) was used as a size marker. PCR products obtained from the B. cereus group reference strains were purified using the AMPure system (Agencourt Bioscience Corporation) following the manufacturer's instructions and direct sequencing was performed with the PCR primers and 50 ng of the PCR product (Genomics Services Unit, Health Protection Agency, London). 2.4. Pyrosequencing assay A multiple alignment was performed on the sequence data obtained from the B. cereus group reference strains using the ClustalW algorithm in Bioedit v7.0.4.1 (Hall, 1999) and pyrosequencing primers (5′-AAA TAA AGT AAT TCT T-3′, 5′-AAA TAA ATT AAT TCT T-3′ and 5′-AAG TGT AGT AAT TCT T-3′) complementary to the nucleotide sequence 1 bp after the indel region on the sense strand were designed based on the consensus sequence obtained from the alignment. Pyrosequencing was performed on the PCR amplicons obtained from the strains listed in Table 1. For the pyrosequencing reaction, 20 μL of the biotinylated PCR amplicons were immobilised onto streptavidin coated magnetic beads and denatured to produce single stranded DNA using the PSQ 96 sample prep tool (Qiagen) following the manufacturer's instructions. The immobilised DNA was mixed with 45 μL of 0.3 μM of each pyrosequencing primer in annealing buffer and heated at 80 °C for 10 min to allow primer annealing. The pyrosequencing reaction was performed with the following nucleotide dispensation order: CGT ATG TAG TAT CAG CTG TAT ATG AGA on a PSQ 96MA pyrosequencing instrument (Qiagen). The DNA sequence of a partial region of the indel was obtained from the resulting pyrograms. 3. Results and discussion 3.1. Identification of a B. anthracis specific indel and in silico analysis to determine its specificity The conserved indels in protein sequences provide an important class of molecular markers for evolutionary, biochemical, genetic, systematic and diagnostic studies (Gupta and Griffiths, 2002; Gao and Gupta, 2005). When a conserved indel of a defined length is present at a specific location within a given gene/protein in a well-defined group of species, the most likely explanation is that the genetic change responsible for this rare genetic change occurred in a common ancestor of the group of species and then it was passed on to various descendents. Thus, due to their shared derived ancestry, the conserved indels represent an important class of synapomorphies that can be used to identify and circumscribe different clades of organisms in molecular terms (Gupta, 2009). The extensive work on conserved indels in recent years has revealed that they are present at various phylogenetic depths thus providing a molecular means to identify different taxonomic clades in molecular terms (Griffiths et al., 2005; Gupta and Lorenzini, 2007; Gupta and Mok, 2007; Gao et al., 2009; Gupta, 2009; Gupta and Gao, 2009). Additionally, due to their specificity for particular groups, they also have the potential to be used as diagnostic markers (Griffiths et al., 2005; Gupta and Griffiths, 2006). The genus Bacillus represents a heterogeneous group of species whose defining feature is the production of endospores in the presence of oxygen (Fritze, 2004; Blackwood et al., 2004). Several studies have shown that the species that are presently part of this genus do

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Table 2 List of complete and unfinished genomes available on Genbank belonging to the B. cereus group and the corresponding Genbank accession number for the YeaC protein and GI number for the YeaC gene.

Complete genomes

Unfinished genomes

Genome

YeaC gene

Organism

Genome Genbank acccession

Protein Genbank accession

Nucleotide GI number

Presence or absence of the YeaC deletion

Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus 27 Bacillus Bacillus

anthracis str. Ames Ancestor anthracis str. A0248 anthracis str. Ames anthracis str. CDC 684 anthracis str. Sterne cereus 03BB102 cereus AH187 cereus AH820 cereus ATCC 10987 cereus ATCC 14579T cereus B4264 cereus E33L cereus G9842 cereus Q1 cereus biovar anthracis str. CI thuringiensis BMB171 thuringiensis serovar konkukian str. 97-

AE017334 CP001598 AE016879 CP001215 AE017225 CP001407 CP001177 CP001283 AE017194 AE016877 CP001176 CP000001 CP001186 CP000227 CP001746 CP001903 AE017355

NP_844520 YP_002866500 NP_844520 YP_002815067 YP_028237 YP_002749484 YP_002338227 YP_002451107 NP_978517 NP_831883 YP_002366851 YP_083521 YP_002445540 YP_002529816 YP_003791877 YP_003664435 YP_036282

GI:30262143 GI:229599883 GI:30260195 GI:227812678 GI:49183039 GI:225862057 GI:217957581 GI:218901206 GI:42779081 GI:30018278 GI:218230750 GI:52140164 GI:218895141 GI:222093774 GI:301051741 GI:296500838 GI:49476684

+ + + + + − − − − − − − − − − − −

thuringiensis str. Al Hakam weihenstephanensis KBAB4

CP000485 CP000903

YP_894710 YP_001644825

GI:118475778 GI:163938013

− −

AAEO00000000 AAEQ00000000 AAEP00000000 AAER00000000

ZP_05182734 ZP_05200487 ZP_05203508 ZP_05194860

GI:48248975 GI:311703261 GI:48242162 GI:311703296

− + + +

AAEK00000000

ZP_00237611

GI:47566775

−

Bacillus anthracis str. A1055 Bacillus anthracis str. Kruger B Bacillus anthracis str. Vollum Bacillus anthracis str. Western North America USA6153 Bacillus cereus G9241

not form a monophyletic lineage (Blackwood et al., 2004; Fritze, 2004). Hence, the present study, which led to the discovery of the large indel that is specific for B. anthracis, was undertaken with the objective of identifying conserved indels that are specific for either all Bacillus species or different subgroups of them that could prove useful in understanding/revising the taxonomy of this genus. Although this work has led to identification of many conserved indels that are specific for a number of subclades of Bacillus species (unpublished results), these indels are not of direct relevance to the present work. However, during the present study, we discovered that one of the proteins, YeaC that was conserved in all sequenced Bacillus species, contained a 24 aa deletion in a conserved region that was uniquely present in B. anthracis. A partial sequence alignment of the

YeaC protein from a selection of species belonging to the phylum Firmicutes for the region where this indel is present is shown in Fig. 1. As seen, this large gap is uniquely present in B. anthracis and not found in any other Bacillus or Firmicutes species that were part of this sequence alignment. The absence of this sequence gap in other Bacillus or Firmicutes species indicates that this indel represents a deletion in B. anthracis rather than an insertion in other groups. The yeaC gene encodes for a hypothetical protein whose putative function is described as a MoxR-like ATPase (Borriss et al., 1996). However, the cellular functions of the MoxR family ATPases are poorly understood although they are thought to play a chaperone-like role in the assembly and activation of specific protein complexes (Snider and Houry, 2006). The large indel in the YeaC protein was the only B. anthracis-specific

Fig. 1. A partial region of an alignment of YeaC protein sequences derived from a selection of species belonging to the phylum Firmicutes whose genome sequences were available. Protein sequences are listed by the species name and corresponding Genbank Accession. The B. anthracis sequence containing a 24 amino acid indel is highlighted in red and the amino acid positions marked according to the numbering of the B. subtilis subsp. subtilis str. 168.

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indel that was identified in this study, however, this does not preclude the existence of other conserved indels that are specific for this species. Examination of the protein sequences from all completed genomes and a selection of unfinished genomes belonging to the B. cereus group (see Table 2) that were available indicated the indel in the YeaC protein was specific to B. anthracis. The only exception was that the homologous protein from the genome of B. anthracis A1055 did not contain this deletion. This strain contains only the pX02 plasmid and is a member of B. anthracis group C, a rare genetic lineage from which only two cases of Anthrax are known (Pearson et al., 2004). Currently no additional information concerning the relationship of this strain to other B. anthracis is available. Hence, it is possible that this strain is either very distantly related to the other B. anthracis strains or it is misclassified as B. anthracis as the parameters used to circumscribe the species are not irrefutable. The close genetic interrelationship between B. anthracis, B. cereus and B. thuringiensis and difficulties in distinguishing between them has been well documented (Chen and Tsen, 2002; Daffonchio et al., 2000; Helgason et al., 2000, 2004; Ticknor et al., 2001). A number of MLST studies using various combinations of genes have shown that B. anthracis is a monophyletic clade (Priest et al., 2004). However, a number of atypically virulent species have been found to exhibit close genetic relationship to the B. anthracis lineage compared with other members of the B. cereus group (Klee et al., 2010). A number of interesting genome sequences from members of the B. cereus group are currently available, that allow determination of the specificity of this indel. Han et al. (2006) sequenced the genomes of two atypical isolates (B. thuringiensis serovar konkukian str. 97–27 and B. cereus E33L) that are more closely related to B. anthracis than to B. cereus or B. thuringiensis by amplified fragment length polymorphism. Both these strains lacked the B. anthracis specific deletion in the YeaC protein (Table 2). B. cereus G9241 is another strain which was found to be very closely related to B. anthracis based on MLST and genomic analysis (Hoffmaster et al., 2006). Interestingly this strain was found to contain a plasmid (pBCX01) sharing high sequence similarity and synteny with pX01 and also encoding for the anthrax toxin. The YeaC protein from the genome of this strain also lacked the B. anthracis-specific deletion (Table 2). Klee et al. (2010) sequenced the genome of a Bacillus strain (B. cereus var. anthracis strain CI) that induced lethal anthrax in a chimpanzee. This strain harboured 3 plasmids, 2 of which shared N99% identity and a high degree of synteny with pX01 and pX02. However, MLST found that this strain did not cluster with the B. anthracis lineage exclusively but rather was interspersed with B. anthracis and the closely related strain B. thuringiensis serovar konkukian str. 97–27 (Klee et al., 2010). This isolate represented the first known non-B. anthracis member of the B. cereus group to possess both virulence plasmids and screening of its genome sequence found that the 24 amino acid deletion was also not present in this strain. These observations reinforce the ability of the YeaC deletion to accurately distinguish B. anthracis from near neighbour virulent species. The identification of B. anthracis often relies on the detection of virulence genes encoded on plasmids. Common targets on pX01 include the genes that encode for anthrax toxin proteins cya (edema factor), lef (lethal factor), pagA (protective antigen) and genes often targeted on pX02 include capA, capB and capC which are essential for polyglutamate capsule synthesis (Bell et al., 2002; Ellerbrok et al., 2002; Fasanella et al., 2003; Hadjinicolaou et al., 2009). The advantage of using these plasmid-encoded genes is that their detection gives an indication of virulence. However, there are also several drawbacks when using these gene targets for identification of B. anthracis. Plasmids can be lost from the organism making it difficult to identify avirulent strains of B. anthracis. The loss of plasmids has been reported from B. anthracis both in nature and in the laboratory and the loss of pX02 in environmental strains is more common than

that of pX01 (Mock and Fouet, 2001; Rao et al., 2010). In addition, near neighbour species that harbour plasmids closely related to pX01 and pX02 can yield false positive results leading to their misidentification (Hoffmaster et al., 2006; Klee et al., 2010). 3.2. PCR assay to examine the presence of the YeaC indel from a panel of Bacillus reference strains Identification of the large indel in the yeaC gene that is specifically present in different strains of B. anthracis but not found in various strains of B. cereus group including those that have been reported to cause an anthrax like disease suggest that this indel could serve as a valuable diagnostic marker to distinguish B. anthracis from other closely related species. Further, unlike the plasmid based markers that can be lost or laterally transferred, the chromosomal location of the yeaC gene offers the advantage that this target gene should not be readily lost or transferred to other species, thus enabling detection of B. anthracis regardless of a strain's virulence. Hence, studies were undertaken to evaluate the performance of this molecular marker for the identification of B. anthracis by different means. A PCR assay was initially developed to amplify the target region from B. cereus group of species and sequencing by conventional methods. To validate the specificity of the B. anthracis indel and of the PCR assay, a selection of reference strains from members of the genus Bacillus were used. 21 reference strains belonging to the B. cereus group that are listed in Table 1 were tested and 20/21 (all except B. weihenstephanensis DSM 11821) gave a positive PCR product of the predicted size (~282–354 bp). These fragments were subsequently sequenced by conventional means. Agarose gel electrophoresis of the obtained PCR products showed a difference in the size between the PCR products obtained from B. anthracis reference strains (fragment size 282 bp) and other reference strains belonging to the B. cereus group (fragment size 354 bp) (Fig. 2). The resulting PCR fragments from these two groups were readily distinguished by means of agarose gel electrophoresis (Fig. 2). The specificity of the PCR assay for members of the B. cereus group was determined by testing the assay against a panel of non-B. cereus group reference strains (listed in Table 1). Using the B. cereus group-specific primer sets that were employed, no PCR amplification was observed with any of the nonB. cereus group reference strains that were tested. These results indicate that the PCR assay is highly specific for members of the B. cereus group (B. cereus, B. thuringiensis and B. anthracis) and it is able to

1

2

3

4

5

Fig. 2. A 2% agarose gel of PCR amplicons from B. cereus NCTC 8035 (lane 1), B. cereus NCTC 11145 (lane 2), B. anthracis NCTC 7753 (lane 4) and B. anthracis NCTC 7752 (lane 5). A 100 bp ladder was run on lane 3 with the brightest band representing the 500 bp fragment.

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Fig. 3. A partial region of the nucleotide alignment encompassing the indel region from all reference strains. The complementary sequence of the pyrosequencing primer is enclosed in the boxed region on the alignment and the primer used to synthesise the DNA sequence of the antisense strand across the indel region.

readily distinguish B. anthracis from B. cereus and B. thuringiensis simply on the basis of the size of the PCR products. Although amplification was not achieved from B. weihenstephanensis DSM 11821 this species is more distantly related to B. anthracis and is not as problematic to distinguish from B. anthracis as the more closely related species B. cereus and B. thuringiensis. Sequencing of the PCR amplicons revealed that all B. anthracis reference strains contained a 72 bp deletion corresponding to the 24 amino acid indel and this deletion was not present in any other reference strains belonging to the B. cereus group. Fig. 3 shows a partial region of the nucleotide alignment of the sequenced PCR products that contain the B. anthracis specific deletion. This indel thus represents a conserved trait that can be used for distinguishing isolates of B. anthracis from other closely related species within the B. cereus group. 3.3. Pyrosequencing assay for B. anthracis based upon the yeaC indel Pyrosequencing technology provides a rapid, simple and inexpensive alternative to conventional sequencing and is particularly useful in microbial diagnostics where short signature sequences or SNPs for a particular organism can be targeted and results obtained in real time (Ronaghi, 2001; Slinger et al., 2007; Wahab et al., 2005). In

view of the specificity of the yeaC indel for B. anthracis, a pyrosequencing assay was developed that targets a partial region of the DNA sequence across this indel region as a rapid alternative method to conventional sequencing. The primer pair used for PCR and direct sequencing included one biotinylated primer (BSU06330_F) that resulted in the PCR amplicons containing a biotin label at the 5′ end of the sense strand in the amplicon. A pyrosequencing primer was designed that was complementary to the plus strand and annealed at the 3′ end of the indel region to allow for DNA synthesis of the antisense strand from 1 base position flanking the 3′ end of the indel (the primer annealing region is shown on Fig. 3). The nucleotide dispensation order for the pyrosequencing reaction incorporated the first 29 bp of the indel's 72 bp DNA sequence in the non B. anthracis reference strains. Positive PCR amplifications were subjected to pyrosequencing, however 3 reference strains (B. mycoides NCTC 9680/NCTC 7586 and B. pseudomycoides DSM 12442) were not successfully pyrosequenced. An example of the pyrograms obtained from a B. anthracis reference strain (NCTC 7753) and a B. cereus reference strain (NCTC 8035) are shown in Fig. 4. Based on the large differences between the two pyrograms, these two species can be clearly distinguished. Sequence data derived from the reference strains using the pyrosequencing assay are provided in Sup. Table 1.

Fig. 4. An example of a B. anthracis and B. cereus pyrogram. B. anthracis NCTC 7753 and B. cereus NCTC 8035 were used as examples. The initial addition of enzyme (E) and substrate (S) followed by the sequential addition of nucleotides are shown on the X-axis. The Y-axis is a measure of the amount of light (relative light units) produced as a result of the pyrophosphate released when a nucleotide is incorporated into the strand being synthesised. Incorporation of identical consecutive nucleotides gave rise to peaks proportionally higher than when one nucleotide was incorporated.

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The pyrosequencing data for these strains matched the sequence data obtained by conventional sequencing confirming the robustness of the pyrosequencing assay. The pyrosequencing data shows that the sequence obtained from the B. anthracis reference strains are conserved. The B. anthracis specific sequence in all cases was deduced as ‘CTTCTGGTG’ which can be clearly distinguished from the non-B. anthracis sequence data. The pyrosequencing assay was then employed against a larger panel of strains that included food and environmental isolates presumptively identified as belonging to the B. cereus group by 16S rRNA sequence analysis and also B. anthracis strains from the anthrax strain collection (ASC). A total of 81 strains were tested using the pyrosequencing assay and the results are shown in Sup. Table 1. A total of 79/81 were amplified and sequenced successfully. All B. anthracis strains provided by the ASC were found to contain the speciesspecific sequence ‘CTTCTGGTG’ confirming that these strains belong to B. anthracis. All food and environmental strains produced pyrograms whose sequence matched those obtained from the non-B. anthracis reference strains indicating that they do not correspond to B. anthracis. The sequence results for the non-B. anthracis strains showed that within the indel sequence considerable sequence heterogeneity was observed between these species/strains. However, further inspection of the sequence data indicated that all non-B. anthracis strains contain a conserved ‘A’ at the third base position in the sequence obtained from the pyrograms, whereas a conserved ‘T’ was present in this position in all B. anthracis strains. Thus, based upon this consistent difference, it is possible to distinguish B. anthracis from other members of the B. cereus group within the first 3 synthesised nucleotides, whereby if the third nucleotide is ‘T’ it belongs to B. anthracis and if it is an ‘A’ the strain would be characterised as a non-B. anthracis member of the B. cereus group. This assay would require less time for the pyrosequencing reaction and therefore less time to obtain the result. Thus, the pyrosequencing assay described here provides a robust and rapid method for the identification of B. anthracis and it is able to distinguish B. anthracis from the closely related species B. cereus and B. thuringiensis. This identification was achieved regardless of the strains plasmid content and thus it is able to distinguish B. anthracis independent of its virulence. The only exception observed was the absence of this indel in the sequence of the YeaC protein from the genome of B. anthracis A1055. Currently, very little information is available concerning the relationship of this strain to other B. anthracis strains but the exact status of this strain remains unclear. 4. Conclusion The B. anthracis specific-indel described in this study provides a robust and highly specific molecular marker for distinguishing B. anthracis from other closely related B. cereus group of species. Due to the large size of this indel, the distinction between B. anthracis and other B. cereus group of species can be readily made by the differences in the size of the PCR-amplified fragments, which can be very simply detected by means of agarose gel electrophoresis, without the need for DNA sequencing. We have also translated this indel to develop a pyrosequencing assay that offers a rapid and unambiguous method for the identification of B. anthracis. This method can also be used to simultaneously screen up to 96 samples presumptively identified as belonging to the B. cereus group to determine if B. anthracis is present or not. Lastly, although the conserved indels in gene/protein sequences have been widely used to identify and distinguish higher taxonomic clades of bacteria, the present work demonstrates that this approach is equally applicable for identification of organisms down to species/subspecies levels and for developing sensitive and specific means for identification of important human pathogens. Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mimet.2011.08.015.

Acknowledgements This work was part of a PhD thesis entitled ‘Elucidating the complex phylogeny of the genus Bacillus’ by author N. Z Ahmod (Sept 2009). The author gratefully acknowledges the award of an HPA PhD studentship to undertake this work and, the HPA's Foreign Travel Committee, for a Travel Fellowship to spend 6 weeks in the laboratory of co-author R. S Gupta. The authors would like to thank Dr C. Arnold and Dr. L Cross from the HPA's Department for Bioanalysis and Horizon Technologies for providing advice on the pyrosequencing aspect of the work and, Dr R. Ellis (Veterinary Laboratories Agency, Weybridge) for providing environmental strains belonging to the B. cereus group and The Special Pathogens Reference Unit, Health Protection Agency, Porton Down for providing B. anthracis DNA. References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Ash, C., Farrow, J.A.E., Dorsch, M., Stackebrandt, E., Collins, M.D., 1991. Comparative analysis of Bacillus anthracis, Bacillus cereus, and related species on the basis of reverse transcriptase sequencing of 16S rRNA. Int. J. Syst. Bacteriol. 41, 343–346. Barker, M., Thakker, B., Priest, F.G., 2005. Multilocus sequence typing reveals that Bacillus cereus strains isolated from clinical infections have distinct phylogenetic origins. FEMS Microbiol. Lett. 245, 179–184. Bavykin, S.G., Yuri, L.P., Vladimir, Z., Kelly, J.J., Jackman, J., Stahl, D.A., Cherni, A., 2004. Use of 16S rRNA, 23S rRNA, and gyrB gene sequence analysis to determine phylogenetic relationships of Bacillus cereus group microorganisms. J. Clin. Microbiol. 42, 3711–3730. Bell, C.A., Uhl, J.R., Hadfield, T.L., David, J.C., Meyer, R.F., Smith, T.F., Cockerill III, F.R., 2002. Detection of Bacillus anthracis DNA by LightCycler PCR. J. Clin. Microbiol. 40, 2897–2902. Blackwood, K.S., Turenne, C.Y., Harmsen, D., Kabani, A.M., 2004. Reassessment of sequence-based targets for identification of Bacillus species. J. Clin. Microbiol. 42, 1626–1630. Borriss, R., Porwollik, S., Schroeter, R., 1996. The 52°–55° segment of the Bacillus subtilis chromosome: a region devoted to purine uptake and metabolism, and containing the genes cotA, gabP and guaA and the pur gene cluster within a 34960 bp nucleotide sequence. Microbiology 142, 3027–3031. Carlson, C.R., Caugant, D.A., Kolstø, A., 1994. Genotypic diversity among Bacillus cereus and Bacillus thuringiensis strains. Appl. Environ. Microbiol. 60, 1719–1725. Chen, M.L., Tsen, H.Y., 2002. Discrimination of Bacillus cereus and Bacillus thuringiensis with 16S rRNA and gyrB gene based PCR primers and sequencing of their annealing sites. J. Appl. Microbiol. 92, 912–919. Daffonchio, D., Cherif, A., Borin, S., 2000. Homoduplex and heteroduplex polymorphisms of the amplified ribosomal 16 S–23 S internal transcribed spacers describe genetic relationships in the “Bacillus cereus group”. Appl. Environ. Microbiol. 66, 5460–5468. Drobniewski, F.A., 1993. Bacillus cereus and related species. Clin. Microbiol. Rev. 6, 324–338. Ellerbrok, H., Nattermann, H., Ozel, M., Beutin, L., Appel, B., Pauli, G., 2002. Rapid andsensitive identification of pathogenic and apathogenic Bacillus anthracis by realtime PCR. FEMS Microbiol. Lett. 214, 51–59. Fasanella, A., Losito, S., Adone, R., Ciuchini, F., Trotta, T., Altamura, S.A., Chiocco, D., Ippolito, G., 2003. PCR assay to detect Bacillus anthracis spores in heat-treated specimens. J. Clin. Microbiol. 41, 896–899. Fritze, D., 2004. Taxonomy of the genus Bacillus and related genera: the aerobic endospore-forming bacteria. Phytopathology 94, 1245–1248. Gao, B., Gupta, R.S., 2005. Conserved indels in protein sequences that are characteristic of the phylum Actinobacteria. Int. J. Syst. Evol. Microbiol. 55, 2401–2412. Gao, B., Mohan, R., Gupta, R.S., 2009. Phylogenomics and protein signatures elucidating the evolutionary relationships among the Gammaproteobacteria. Int. J. Syst. Evol. Microbiol. 59, 234–247. Griffiths, E., Petrich, A.K., Gupta, R.S., 2005. Conserved indels in essential proteins that are distinctive characteristics of Chlamydiales and provide novel means for their identification. Microbiology 151, 2647–2657. Gupta, R.S., 2009. Protein signatures (molecular synapomorphies) that are distinctive characteristics of the major cyanobacterial clades. Int. J. Syst. Evol. Microbiol. 59, 2510–2526. Gupta, R.S., Gao, B., 2009. Phylogenomic analyses of clostridia and identification of novel protein signatures that are specific to the genus Clostridium sensu stricto (cluster I). Int. J. Syst. Evol. Microbiol. 59, 285–294. Gupta, R.S., Griffiths, E., 2002. Critical issues in bacterial phylogeny. Theor. Popul. Biol. 61, 423–434. Gupta, R.S., Griffiths, E., 2006. Chlamydiae-specific proteins and indels: novel tools for studies. Trends Microbiol. 14, 527–535. Gupta, R.S., Lorenzini, E., 2007. Phylogeny and molecular signatures (conserved proteins and indels) that are specific for the Bacteroidetes and Chlorobi species. BMC Evol. Microbiol. 7, 71–89. Gupta, R.S., Mok, A., 2007. Phylogenomics and signature proteins for the alpha Proteobacteria and its main groups. BMC Microbiol. 7, 106–126.

N.Z. Ahmod et al. / Journal of Microbiological Methods 87 (2011) 278–285 Hadjinicolaou, A.V., Demetriou, V.L., Hezka, J., Beyer, W., Hadfield, T.L., Kostrikis, L.G., 2009. Use of molecular beacons and multi-allelic real-time PCR for detection of and discrimination between virulent Bacillus anthracis and other Bacillus isolates. J. Microbiol. Methods 78, 45–53. Hall, T.A., 1999. Bioedit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95–98. Han, C.S., Xie, G., Challacombe, J.F., Altherr, M.R., Bhotika, S.S., Brown, N., Bruce, D., Campbell, C.S., Campbell, M.L., Chen, J., Chertkov, O., Cleland, C., Dimitrijevic, M., Doggett, N.A., Fawcett, J.J., Glavina, T., Goodwin, L.A., Green, L.D., Hill, K.K., Hitchcock, P., Jackson, P.J., Keim, P., Kewalramani, A.R., Longmire, J., Lucas, S., Malfatti, S., McMurry, K., Meincke, L.J., Misra, M., Moseman, B.L., Mundt, M., Munk, A.C., Okinaka, R.T., Parson-Quintana, B., Reilly, L.P., Richardson, P., Robinson, D.L., Rubin, E., Saunders, E., Tapia, R., Tesmer, J.G., Thayer, N., Thompson, L.S., Tice, H., Ticknor, L.O., Wills, P.L., Brettin, T.S., Gilna, P., 2006. Pathogenomic sequence analysis of Bacillus cereus and Bacillus thuringiensis isolates closely related to Bacillus anthracis. J. Bacteriol. 188, 3382–3390. Hansen, B.M., Hendriksen, N.B., 2001. Detection of enterotoxic Bacillus cereus and Bacillus thuringiensis strains by PCR analysis. Appl. Environ. Microbiol. 67, 185–189. Helgason, E., Okstad, O.A., Caugant, D.A., Johansen, H.A., Fouet, A., Mock, M., Hegna, I., Kolstø, A.B., 2000. Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis—one species on the basis of genetic evidence. Appl. Environ. Microbiol. 66, 2627–2630. Helgason, E., Tourasse, N.J., Meisal, R., Caugant, D.A., Kolstø, A., 2004. Multilocus sequence typing scheme for bacteria of the Bacillus cereus group. Appl. Environ. Microbiol. 70, 191–201. Hoffmaster, A.R., Hill, K.K., Gee, J.E., Marston, C.K., De, B.K., Popovic, T., Sue, D., Wilkins, P.P., Avashia, S.B., Drumgoole, R., Helma, C.H., Ticknor, L.O., Okinaka, R.T., Jackson, P.J., 2006. Characterization of Bacillus cereus isolates associated with fatal pneumonias: strains are closely related to Bacillus anthracis and harbor B. anthracis virulence genes. J. Clin. Microbiol. 44, 3352–3360. Hurtle, W., Bode, E., Kulesh, D.A., Kaplan, R.S., Garrison, J., Bridge, D., House, M., Frye, M.S., Loveless, B., Norwood, D., 2004. Detection of the Bacillus anthracis gyrA gene by using a minor groove binder probe. J. Clin. Microbiol. 42, 179–185. Jensen, G.B., Hansen, B.M., Eilenberg, J., Mahillon, J., 2003. The hidden lifestyles of Bacillus cereus and relatives. Environ. Microbiol. 5, 631–640. Klee, S.R., Brzuszkiewicz, E.B., Nattermann, H., Brüggemann, H., Dupke, S., Wollherr, A., Franz, T., Pauli, G., Appel, B., Liebl, W., Couacy-Hymann, E., Boesch, C., Meyer, F., Leendertz, F.H., Ellerbrok, H., Gottschalk, G., Grunow, R., Liesegang, H., 2010. The genome of a Bacillus isolate causing anthrax in chimpanzees combines chromosomal properties of B. cereus with B. anthracis virulence plasmids. PLoS One 5, e10986. Ko, K.S., Kim, J.M., Kim, J.W., Jung, B.Y., Kim, W., Kim, I.J., Kook, Y.H., 2003. Identification of Bacillus anthracis by rpoB sequence analysis and multiplex PCR. J. Clin. Microbiol. 41, 2908–2914. Kolstø, A.B., Tourasse, N.J., Okstad, O.A., 2009. What sets Bacillus anthracis apart from other Bacillus species? Annu. Rev. Microbiol. 63, 451–476. La Duc, M.T., Satomi, M., Agata, N., Venkateswaran, K., 2004. GyrB as a phylogenetic discriminator for the members of the Bacillus anthracis-cereus-thuringiensis group. J. Microbiol. Methods 56, 383–394. Lechner, S., Mayr, R., Francis, K.P., Prüß, B.M., Kaplan, T., Wießner-Gunkel, E., Stewart, G.S.A.B., Scherer, S., 1998. Bacillus weihenstephanensis sp. nov. is a new psychrotolerant species of the Bacillus cereus group. Int. J. Syst. Evol. Microbiol. 48, 1373–1382. Mock, M., Fouet, A., 2001. Anthrax. Annu. Rev. Microbiol. 55, 647–671. Nakamura, L.K., Jackson, M.A., 1995. Clarification of the taxonomy of Bacillus mycoides. Int. J. Syst. Bacteriol. 45, 46–49. Oggioni, M.R., Meacci, F., Carattoli, A., Ciervo, A., Orru, G., Cassone, A., Pozzi, G., 2002. Protocol for real-time PCR identification of anthrax spores from nasal swabs after broth enrichment. J. Clin. Microbiol. 40, 3956–3963. Pearson, T., Busch, J.D., Ravel, J., Read, T.D., Rhoton, S.D., U'Ren, J.M., Simonson, T.S., Kachur, S.M., Leadem, R.R., Cardon, M.L., Ert, M.N., Huynh, L.Y., Fraser, K.M., Keim, P., 2004. Phylogenetic discovery bias in Bacillus anthracis using single-nucleotide polymorphisms from whole-genome sequencing. PNAS 101, 13536–13541. Priest, F.G., Barker, M., Baillie, L.W.J., Holmes, E.C., Maiden, M.C.J., 2004. Population structure and evolution of the Bacillus cereus group. J. Bacteriol. 186, 7959–7970. Qi, Y., Patra, G., Liang, X., Williams, L.E., Rose, S., Redkar, R.J., Delvecchio, V.G., 2001. Utilization of the rpoB gene as a specific chromosomal marker for real-time PCR detection of Bacillus anthracis. Appl. Environ. Microbiol. 67, 3720–3727. Ramisse, V., Patra, G., Vaissaire, J., Mock, M., 1999. The Ba813 chromosomal DNA sequence effectively traces the whole Bacillus anthracis community. J. Appl. Microbiol. 87, 224–228. Rao, S.S., Mohan, K.V., Atreya, C.D., 2010. Detection technologies for Bacillus anthracis: prospects and challenges. J. Microbiol. Methods 82, 1–10. Read, T.D., Peterson, S.N., Tourasse, N., Baillie, L.W., Paulsen, I.T., Nelson, K.E., Tettelin, H., Fouts, D.E., Eisen, J.A., Gill, S.R., Holtzapple, E.K., Okstad, O.A., Helgason, E., Rilstone, J., Wu, M., Kolonay, J.F., Beanan, M.J., Dodson, R.J., Brinkac, L.M., Gwinn, M., DeBoy, R.T., Madpu, R., Daugherty, S.C., Durkin, A.S., Haft, D.H., Nelson, W.C., Peterson, J.D., Pop, M., Khouri, H.M., Radune, D., Benton, J.L., Mahamoud, Y., Jiang, L., Hance, I.R., Weidman, J.F., Berry, K.J., Plaut, R.D., Wolf, A.M., Watkins, K.L., Nierman, W.C., Hazen, A., Cline, R., Redmond, C., Thwaite, J.E., White, O., Salzberg, S.L., Thomason, B., Friedlander, A.M., Koehler, T.M., Hanna, P.C., Kolstø, A.B., Fraser, C.M., 2003. The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria. Nature 423, 81–86. Ronaghi, M., 2001. Pyrosequencing sheds light on DNA sequencing. Genome Res. 11, 3–11.

285

Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D.R., Dean, D.H., 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62, 775–806. Slinger, R., Yan, L., Myers, R., Ramotar, K., St Denis, M., Aaron, S.D., 2007. Pyrosequencing of a recA gene variable region for Burkholderia cepacia complex genomovar identification. Diagn. Microbiol. Infect. Dis. 58, 379–384. Snider, J., Houry, W.A., 2006. MoxR AAA+ ATPases: A novel family of molecular chaperones? J. Struct. Biol. 156, 200–209. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882. Ticknor, L.O., Kolstø, A.B., Hill, K., Keim, P., Laker, M.T., Tonks, M., Jackson, P.J., 2001. Fluorescent amplified fragment length polymorphism analysis of Norwegian Bacillus cereus and Bacillusithuringiensis soil isolates. Appl. Environ. Microbiol. 67, 4863–4873. Wahab, T., Hjalmarsson, S., Wollin, R., Engstrand, L., 2005. Pyrosequencing Bacillus anthracis. Emerg. Infect. Dis. 11.

Glossary Aerobe: An organism able to grow in the presence of atmospheric oxygen. Anthrax: An acute infectious disease of animals including humans caused by the bacterium Bacillus anthracis. Bacteriophage gamma: A type of virus that infects a bacterial cell. Classification: The arrangement of organisms into groups based on similarity or evolutionary relatedness. Chaperone protein: A protein that assists the non-covalent folding or unfolding and the assembly or disassembly of other macromolecular structures. Deoxyribonucleic acid/DNA: The nucleic acid that constitutes the genetic material of all cellular organisms. It is a polynucleotide composed of deoxyribonucleotides connected by phosphodiester bonds. Endospore: An extremely heat and chemical resistant, dormant, thick-walled spore that develops within bacteria. Enterotoxin: A protein toxin released by a microorganism affecting the cells of the intestinal mucosa. Facultative Anaerobe: Microorganisms that do not require oxygen for growth, but grow better in its presence. Genome: All the genetic material of a particular organism; its size is measured as the total number of base pairs. Gram positive: Species belonging to the phyla Actinobacteria and Firmicutes, which have a single membrane and a thick cell wall made of crosslinked peptidoglycan. Hypothetical protein: A protein whose presence has been predicted, but has no experimental evidence for its expression. Indel: Amino acid insertions or deletions in highly conserved proteins that are specific to particular groups of organisms. Microbial diagnostics: The detection and identification of the causative agent of a microbial infection. Microbial systematics: The study of microbial taxonomy and evolutionary relationships. Nosocomial infection: A patient with an infection acquired in a hospital or other healthcare facility. Opportunistic pathogen: An infection caused by a microorganism that does not usually cause disease in a healthy host but can infect a host with a compromised immune system. Pathogen: An organism that can cause disease. Phylogeny: The grouping of species based on evolutionary relationships. Plasmid: Extrachromosomal genetic element of bacteria that replicate independently of the chromosome and can be passed between different bacteria. Polymerase chain reaction/PCR: A laboratory technique used to produce multiple copies of short segments of DNA. Primer: A short polynucleotide chain to which new deoxyribonucleotides can be added by DNA polymerase. Psychotolerant: An organism able to grow at low temperature but with a growth temperature optimum greater than 15 °C. Pyrosequencing: A DNA sequencing technique that relies on detection of pyrophosphate which is released on nucleotide incorporation. Reference strain: A characterised strain of known species identity that serves as the reference for a particular organism. Single nucleotide polymorphism/SNP: A type of DNA polymorphism that occurs when a single nucleotide in the genome sequence is altered. Species: A collection of genetically closely related strains sufficiently different from all other strains to be characterised as a distinct unit. Spores: A dormant, reproductive structure that is adapted for surviving in unfavorable conditions. Toxin: A poisonous substance produced by an organism that is capable of inducing host damage. Virulence factor: A molecule produced by a pathogen that aids its ability to be pathogenic. YeaC protein: Hypothetical protein whose putative function is described as a MoxR like ATPase.