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The Bacillus cereus group includes Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides and Bacillus weihenstephanensis. The small acid-soluble spore protein (SASP) β has been previously demonstrated to be among the biomarkers differentiating B. anthracis and B. cereus; SASP β of B. cereus most commonly exhibits one or two amino acid substitutions when compared to B. anthracis. SASP α is conserved in sequence among these two species. Neither SASP α nor β for B. thuringiensis, B. mycoides and B. weihenstephanensis have been previously characterized as taxonomic discriminators. In the current work molecular weight (MW) variation of these SASPs were determined by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) for representative strains of the 5 species within the B. cereus group. The measured MWs also correlate with calculated MWs of translated amino acid sequences generated from whole genome sequencing projects. SASP α and β demonstrated consistent MW among B. cereus, B. thuringiensis, and B. mycoides strains (group 1). However B. mycoides (group 2) and B. weihenstephanensis SASP α and β were quite distinct making them unique among the B. cereus group. Limited sequence changes were observed in SASP α (at most 3 substitutions and 2 deletions) indicating it is a more conserved protein than SASP β (up to 6 substitutions and a deletion). Another even more conserved SASP, SASP α-β type, was described here for the first time.
The Bacillus cereus group consists of Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides and Bacillus weihenstephanensis. As is well known, B. anthracis is the causative agent of anthrax. Some B. cereus strains induce severe pneumonia  while others are responsible for food-borne gastroenteritis and emesis  as well as opportunistic infections, particularly of the eye . B. thuringiensis (an insect pathogen) and B. mycoides (a soil organism) do not generally cause human disease. Some strains of B. weihenstephanensis cause emesis like B. cereus . Thus it is important that biomarkers be defined for distinguishing B. weihenstephanensis from other members of the B. cereus group. In the food industry contamination commonly occurs with B. cereus, B. mycoides and B. weihenstephanensis so it is also vital to discriminate among two species that have potential for causing human disease (B. cereus and B. weihenstephanensis) and an environmental contaminant (B. mycoides). The particular focus of the current work is the use of SASPs for characterization of B. weihenstephanensis and B. mycoides.
The most studied members of the B. cereus group are B. anthracis, B. cereus and B. thuringiensis which have recently been referred to as the ACT group. There is high genetic similarity among certain members of the B. cereus group (B. anthracis, B. cereus and B. thuringiensis) with B. anthracis being a distinct lineage [5–7]. All share a significant degree of genetic similarity as demonstrated by DNA-DNA hybridization  and 16S and 23S rRNA sequence [9,10]. B. anthracis is generally characterized by the presence of the virulence plasmids, pXO1 and pXO2 which code for the tri-partite toxins and the poly-D-glutamic acid capsule respectively. The extra-chromosomal plasmid markers can be lost during culture; and naturally occurring strains without plasmids have been isolated from the environment. Furthermore other species occasionally contain these plasmids [1, 11–13]. Chromosomal markers include the presence of Ba813  or locus tag BA_5345  and single nucleotide substitutions in rpoB . Locus tag BA_5345 appears to be the most unique. Polymorphisms in 16S rRNA have been used in discrimination of B. anthracis from other members of the B. cereus group . Tandem repeats such as are found in the vrrA gene  and in the bclA gene  are used for strain not species discrimination. A distinguishing characteristic of B. thuringiensis is its ability to produce insecticidal protein crystals (parasporal bodies) but loss of this trait makes B. thuringiensis difficult to distinguish from B. cereus by other physiological or morphological traits.
Discrimination of B. mycoides requires further examination. B. mycoides on initial isolation displays rhizoid colony morphology unlike other members of the B. cereus group. Unfortunately loss of the ability to form mycoidal colonies during laboratory culture is often observed, thus this is not a reliable phenotypic feature . Furthermore even newly isolated strains of B. mycoides may lack this property . Multi-locus enzyme electrophoresis and restriction fragment length polymorphism analyses of B. mycoides show two genetically distinct groups of strains . This was confirmed by others using DNA-DNA hybridization and fatty acid profiling . Subsequently Nakamura, 1998 named these two groups B. mycoides (equivalent to group 1) and B. pseudomycoides (equivalent to group 2). The same fatty acids are present in the ACT group and B. mycoides with only differences in relative levels providing discrimination [22,23]. In addition, 16S rRNA sequencing suggested that one group of B. mycoides is essentially indistinguishable from the ACT group although the other contains significant sequence variation and appears quite unique. Nine of 10 strains of B. mycoides studied (including the type strain DSM 2048) contain 4 substitutions within positions 175–193 (E. coli numbering) of the 16S rRNA versus the ACT group. The remaining strain of B. mycoides exhibited only one substitution versus the ACT group . Others also using 16S rRNA sequencing referred to groups A and B where B. mycoides and B. cereus strains are intermixed. B. pseudomycoides was lumped with some B. mycoides strains by 16S rRNA sequence (group A) . Thus in the present report, to avoid confusion, we have chosen to retain the terms B. mycoides groups 1 and 2.
B. weihenstephanensis is the species most recently described and its relatedness to other members of the B. cereus group also requires further examination . Like B. cereus, B. weihenstephanensis strains are known to cause food poisoning and emesis involving enterotoxin production . Unlike other B. cereus group organisms, B. weihenstephanensis are psychrotolerant having optimal growth at approximately 25–35°C. They can be distinguished by their ability to grow at 7°C and below . This is proving importance in food microbiology since there is an increasing demand for ready-to-eat foods and these strains grow at refrigeration temperatures . Sequence differences are found in 16S rRNA, the 23S rRNA, the 16S–23S rRNA spacer region and the cold-shock protein gene cspA .
SASPs were originally discovered by Peter Setlow‘s group in Bacillus subtilis and their functions include protecting the spore DNA from damage by ultra-violet light . However, the function of the analogous proteins in B. anthracis, have not been extensively studied. SASPs were first characterized by determination of MW using matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry by Catherine Fenselau and co-workers  are located in the core region of Bacillus spores and have been previously demonstrated to be biomarkers for differentiating B. anthracis and B. cereus. B. cereus strains have been divided into two clades [27,28] one more closely related to B. anthracis and one more distantly related. Clade 1 can’t be readily distinguished from some B. thuringiensis strains. Most B. thuringiensis strains are grouped with B. cereus clade 2 . SASP β of B. cereus exhibits one or two amino acid substitutions (correlating with clade 1 and 2) being distinct from B. anthracis [29,30]. SASP α does not vary in sequence among these organisms. Hoffmaster et al.  phylogenetically divided B. cereus strains into 10 branches by amplified fragment length polymorphism with Branch F including all B. anthracis strains and pneumonia-causing strains of B. cereus. There are four sub-branches within Branch F, with B. anthracis strains being found within sub-branch F1-B. SASPs of B. cereus strains found in F1-B showed a single amino acid substitution, while strains in the other three sub-branches were more variable generally showing one or two amino acid substitutions .
The SASPs of B. thuringiensis, B. mycoides and B. weihenstephanensis have not been previously characterized. In the current study the MWs of SASP α and β were compared for the 5 B. cereus species. Available genomic sequences were also downloaded for comparison. The primary aim of the study was to determine the utility of SASPs as possible markers for discrimination of B. weihenstephanensis and B. mycoides from other members of the B. cereus group.
All strains used are listed in Table 1: Bacillus anthracis strains were a gift from Stephen Leppla, NIH and previously characterized by carbohydrate profiles using gas chromatography-mass spectrometry [32,33]; this distinguishes B. anthracis from B. cereus and B. thuringiensis. The B. cereus and B. thuringienisis strains were a gift from Paul Jackson and were characterized using amplified fragment length polymorphism . Bacillus weihenstephanensis strains were a gift from Markus Antwerpen and originated from the Weihenstephan Culture Collection of Bacillus cereus group species, Munich Technical University, Freising-Weihenstephan, Germany (WSBC); 5 of the 6 strains are described in . B. mycoides strains were obtained from the Bacillus Genetic Stock Culture Collection (BSGC). Three of the strains are described in  and were deposited in the BSGC by Leonard Peluski (6A11 [98/1883] 6A12 [96/3308] and 6A14 [Gibson 71] and originated from the Food Safety and Microbiology Laboratory, Central Public Health Laboratory, London, United Kingdom, and Niall Logan, Department of Biological Sciences, Glasgow Caledonian University, Glasgow, United Kingdom); 6A13 (NRS-306, ATCC 6463) was characterized by fatty acid profiling and DNA:DNA homology and previously shown to be a member of group 1 . B. mycoides 6A19 and 6A20 are ATCC strains (31101 and 11986 respectively). Strains were kept as stocks at −70°C in glycerol. Bacteria were plated on sheep blood agar and incubated for 24 hr at 37°C to allow primary growth and to test hemolysis and then plated on nutrient agar and grown at room temperature to allow sporulation.
Spores were prepared as previously . Briefly, bacteria were grown on nutrient agar plates, at room temp until sporulation was essentially complete (>95%). Cultures were harvested and spores were centrifuge-washed three times in distilled water. Samples were freeze-thawed to activate autolysis (by freezing overnight then thawing) and incubating at room temp for 1–2 hr generating preparations >99% spores. The degree of sporulation was assessed, by phase contrast microscopy. The spores were harvested by centrifugation, washed three times in distilled water, autoclaved, lyophilized and stored at −70°C until analysis.
1–3 mg of spores was suspended in 1 ml of water, immediately before mass spectrometry analysis. SASPs were selectively extracted using a strong acidic solution of 10% trifluoroacetic acid (TFA) (Sigma Chemical Co., St. Louis, MO) in water. Extraction of SASPs was performed by mixing 1 μl of the spore suspension in water and 1 μl of 10% TFA in water and dried on the MALDI plate. The matrix consisted of 1 μl of α-cyano-4-hydroxy-cinnamic acid (α-CHCA, 10 mg/ml) (Sigma Chemical Co., St. Louis, MO), which was added to the MALDI plate, and allowed to dry. Samples were analyzed in positive ion, linear mode by MALDI TOF MS for the determination of the MW of native intact SASPs using a Bruker Ultraflex 2 (Bruker Daltonics, Billerica, MA).
The measured MWs of SASP α and β, determined using mass spectrometry, are reported here for representative strains of the 5 species within the B. cereus group. Both these proteins have been previously studied for a limited number of strains for B. anthracis and B. cereus [29–31]). The other SASP α-β type described here has not been previously characterized. Three types of α-β SASPs were identified in Genbank searches. The α SASPs, β SASps and α-β type SASPs were not clearly defined by the annotations. All of the identified SASPs were listed as either β or α-β type. Sequence searches using either α or β SASP sequences would identify the α-β SASPs types since the sequences QMKYEIAQEF, RANGSVGGEI, and TKRLVAMAEQ are conserved within all three types of SASPs. The majority of the sequence differences were isolated within the N and C-termini. Once the α-β type was delineated, utilizing the newly identified alpha-beta type sequence, this type of SASP was identified for all strains examined.
For all 6 strains of B. weihenstephanensis, SASP α had the same MW (range 6869.87 to 6874.18) which differed from strains representing the other 5 species (generally range 6832.39 to 6838.71); see Fig 1 and Table 1. SASP β was also unique for 5/6 strains of B. weihenstephanensis (MW range 6523.62 to 6524.73). Although for one strain (WSBC 10201) the measured MW for SASP β differed from the other 5 strains of B. weihenstephanensis and all other strains for the other 4 species studied (see Table 1). It is unclear whether this stain represents a distinct clade within B. weihenstephanensis or is an example of a different B. cereus group species as yet undefined.
One of the 6 B. mycoides strains characterized in the current report (6A13) has been previously defined as a member of group 1 . The SASPs of 5/6 strains (including 6A13) are reported here to be indistinguishable (based on MW of both SASP α and SASP β) from one another and those of the ACT group (see Table 1) suggesting that all 5 strains are members of group 1. The 6th strain (6A19) has distinct measured MWs from the other 5 strains and contains a unique SASP α and β (MW: 6636.92 and 6536.10 respectively) which presumably makes it a member of group 2.
A search of Genbank for members of the B. cereus group (as of June 1, 2009) provided a total of 19 B. anthracis, 44 B. cereus and 17 B. thuringiensis genomes. Only 1 genome was available each for B. weihenstephanensis, and B. pseudomycoides, and 3 for B. mycoides.
A BLAST search using the SASP α sequence (MANQNSSNQL VVPGATAAID QMKYEIAQEF GVQLGADSTA RANGSVGGEI TKRLVAMAEQ SLGGFHK) to search the above genomes determined that all 19 B. anthracis, 31 of 44 B. cereus and 14 of 17 B. thuringiensis contain an identical sequence. Of the remaining strains, 5 B. cereus and the remaining 3 B. thuringiensis strains have a single amino acid substitution at the carboxyl terminus (glutamine to histidine switch). Two B. cereus strains (sub-species cytotoxis and Rock 3–44) contain sequences almost identical to the other 31 B. cereus strains with one or two amino acid substitutions respectively unique to the strains. The remaining 6 B. cereus strains have sequences identical to those found in B. mycoides strain DSM 2048 and B. weihenstephanensis KBAB4. The other 2 B. mycoides strains and B. pseudomycoides contain sequences unique for this group of 3 strains. Table 2 shows the single deposited B. weihenstephanensis sequence (and for other strains sharing this sequence) and the unique B. mycoides and B. pseudomycoides sequences. Only examples are given of the common B. anthracis, B. cereus and B. thuringiensis sequences. The other unusual sequences (B. cereus sub-species cytotoxis and Rock 3–44) are also provided.
A majority of the strains within the ACT group were found to contain a SASP α-β type which is identical: MARNRNSNQL ASHGAQAALD QMKYEIAQEF GVQLGADTSS RANGSVGGEI TKRLVAMAEQ QLGGGYTR. However, 5 B. cereus strains (AH1273, AH603, AH621, and BDRD-ST196) contain a single amino acid substitution (serine to alanine) at position 7 which is identical to the sequence found in B. weihenstephanensis KBAB4 and B. mycoides DSM 2048. The other B. mycoides/B. pseudomycoides sequences have a single amino acid substitution at position 11 (alanine to valine); see Table 3. This SASP α-β type has not been previously detected using MALDI mass spectrometry since it is generally expressed at lower abundance than SASP α, being almost undetectable in many strains. For the ACT group this protein (see Table 3) would have a calculated mass of 7064.77; for B. weihenstephanensis 7064.78 and for B. mycoides and B. pseudomycoides 7085.80. Fig. 2 shows an example of a mass spectrum where the SASP α-β type is present at similar levels to SASP α and one where it is present at lower levels. SASP β is expressed at even higher levels than SASP α in both cases which is typical.
SASP β was found to be more variable in sequence (see Table 4). Using the B. anthracis sequence (MARSTNKLAV PGAESALDQM KYEIAQEFGV QLGADATARA NGSVGGEITK RLVSLAEQQL GGFQK) 8 distinct sequence variations were seen. The 19 B. anthracis strains all contain identical sequences, but only 2 B. cereus and 1 B. thuringiensis share this sequence. Like many other markers SASP β indicates B. anthracis is a unique lineage but does not serve as a totally unique discriminator from B. cereus and B. thuringiensis. Fourteen strains of B. cereus and 6 strains of B. thuringiensis contain a 1 amino acid substitution (phenylalanine to tyrosine) at the carboxyl end of the protein, while 2 B. cereus and 1 B. thuringiensis contain a single amino acid substitution (alanine to serine) at the amino end of the protein in agreement with previous mass spectrometry analysis [29–31]. Fourteen B. cereus and 4 B. thuringiensis strains contain a double substitution which reflects both single amino acid substitutions (also in agreement with previous studies [29–31].
B. weihenstephanensis KBAB4 and B. mycoides DSM 2048 share identical SASP β sequences with 5 strains of B. cereus. These B. cereus strains (AH1273, AH603, AH621, BDRD-ST196 and AH1272) contain the B. weihenstephanensis SASP α sequence as well. Whether these 5 B. cereus strains are psychrolerant and share other features with B. weihenstephanensis remains to be determined. Again B. weihenstephanensis KBAB4 and Bacillus mycoides DSM 2048 contain identical sequences. The calculated MW, from these deposited sequences, are also indistinguishable from the measured MWs for B. weihenstephanensis strains characterized at USC (calculated MW α: 6874.65, calculated MW β: 6527.27). While the other 3 strains (two B. mycoides and one B. pseudomycoides) share a different sequence. The calculated MWs of both SASP α and β for B. pseudomycoides DSM 12442, B. mycoides Rock 1–4 and B. mycoides Rock 3–17 (6636.41 and 6536.28 respectively) are also consistent with measured MWs (see above). Bacillus cereus Rock 3–44 shares the B. mycoides and B. pseudomycoides sequence for SASP β, but has a unique SASP α sequence
B. cereus subsp. cytotoxis NVH 391–98 is unique among members of the ACT group. Each SASP examined contained single amino acid substitutions which are distinct for this organism. Unique single amino acid substitutions were also seen in B. cereus Rock 3–44 (SASP α ), B. thuringiensis IBL 200 (SASP α-β type), and B. cereus AH1271 (SASP β).
In summary, based on MW determined experimentally and the genomic sequences present in Genbank, B. weihenstephanensis can generally be distinguished from the 4 other species of the B. cereus group. Members of the B. mycoides species contain 2 distinct deposited sequences, one of which is identical to B. weihenstephanensis and another which is distinct for the B. mycoides group. The measured MWs also show 2 types; one consistent with the ACT group and the other also distinct for B. mycoides. Further studies on B. mycoides are clearly warranted.
In previous work it was demonstrated that SASP β is useful in discriminating B. anthracis from B. cereus but SASP α is highly conserved [29–31]. In the current work it was confirmed that B. thuringiensis has similar characteristics to B. cereus. However, B. mycoides group 1 is also lumped with the ACT group as regards both SASP α and β. This is consistent with previous reports of the 16S rRNA sequence of B. mycoides group 1 . B. mycoides group 2 (including B. pseudomycoides) and B. weihenstephanensis are quite distinct from the ACT group for both SASP α and β. This is also in agreement with 16S rRNA analysis . The sequences available from genomic analysis by others are in general agreement with the results from mass spectrometry analysis. However, it appears that some strains of B. mycoides are still difficult to distinguish from B. cereus and other strains of B. mycoides can be confused with B. weihenstephanensis. A previously un-described SASP α-β type is described here for the first time that may have utility in aiding discrimination of B. mycoides and B. weihenstephanensis.
In conclusion, food contamination commonly occurs with B. cereus, B. mycoides and B. weihenstephanensis so it essential to discriminate among two species that have potential for causing human disease (B. cereus and B. weihenstephanensis) and an environmental contaminant (B. mycoides). B. weihenstephanensis displays a unique feature (psychrotolerance) usually distinguishing it from other members of the B. cereus group. However, the rhizoidal nature of B. mycoides is not apparent for many strains and regardless is a feature readily lost on culture. Thus there is a particular need for further markers to be discovered for better characterization of both B. mycoides and B. pseudomycoides.
The authors are grateful to Markus Antwerpen for his help in supplying strains of B. weihenstephanensis which were previously not available from any culture collection in the US. Support for this study was provided by the Sloan Foundation Indoor Air Program. Courtney Callahan received fellowships from the Sloan Foundation, NIH (INBRE Program) and the NSF SEAGEP Program. Automated Methods LLC (A. and K. Fox) received a Phase Zero SBIR through the South Carolina Research Authority (SCRA).
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