Short Communication The Bacillus cereus containing sub-branch most closely related to Bacillus anthracis, have single amino acid substitutions in small acid-soluble proteins, while remaining sub-branches are more variable Courtney Callahan * , Elisangela R. Castanha, Karen F. Fox, Alvin Fox Department of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, 6311 Garners Ferry Road, Columbia, SC 29208, USA article info Article history: Received 19 November 2007 Accepted 21 November 2007 Available online 15 February 2008 Keywords: Bacillus cereus Anthrax Small acid-soluble proteins (SASPs) Electrospray ion trap mass spectrometry Matrix assisted laser desorption/ionization mass spectrometry abstract Hoffmaster et al. [Hoffmaster AR, Ravel J, Rasko DA, Chapman GD, Chute MD, Marston CK, et al. Identi- fication of anthrax toxin genes in Bacillus cereus associated with illness resembling inhalation anthrax. Proc Natl Acad Sci U S A 2004;101:8449–54; Hoffmaster AR, Hill KK, Gee JE, Marston CK, De BK, Popovic T, et al. 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 2006;44:3352–60] phylogenetically divided Bacillus cereus strains into 10 branches by amplified fragment length polymor- phism (AFLP) with Branch F including all Bacillus anthracis strains and pneumonia-causing strains of B. cereus. There are four sub-branches within Branch F, referred to here as F1-A, F1-B, F2-A and F2-B. The B. anthracis strains are found within sub-branch F1-B. Concerning, the currently available B. cereus pneumonia-causing isolates, one was found to categorize within sub-branch F1-B and two within F2- B. In the following work the sequence variation between B. cereus strains was determined by MALDI- TOF MS and MS–MS for each strain of B. cereus in Branch F. ESI-MS was performed on selected strains for confirmation. Small acid-soluble proteins (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 single substitutions always occurred in the C-termi- nus. Double substitutions occurred in both N and C termini. Of the pneumonia-causing strains, one ex- hibited a single amino acid substitution, while the other two exhibited a two amino acid substitution. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction There is high genetic similarity among members of the Bacillus cereus group: Bacillus anthracis, B. cereus and Bacillus thuringiensis [1]. B. anthracis is the causative agent of the disease anthrax. Some B. cereus strains can cause severe pneumonia, while others can cause food-borne gastroenteritis and opportunistic infections [2,12,16,17]. B. thuringiensis and the other more distantly related members of the group, Bacillus mycoides and Bacillus weihenstepha- nensis, do not generally cause human disease [14]. La Duc et al. divided B. cereus into those 70% or greater related to B. anthracis and others more distantly related by DNA–DNA homol- ogy [13]. Daffonchio and co-workers also divided B. cereus into two groups of strains by single strand conformation polymorphism [6]. Small acid-soluble proteins (SASPs) are located in the core region of Bacillus spores and have been previously demonstrated as reliable biomarkers for differentiating B. anthracis and B. cereus [9]. B. cereus strains can be lumped into two DNA–DNA homology groups (one >70% related and one more distantly related) to B. anthracis which correlates with whether they exhibit one or two amino acid substi- tutions. The closely related cluster was characterized by a b-SASP with a single amino acid substitution, localized either close to the C-terminus (phenylalanine / tyrosine, 16 masses change) or close to the N terminus (serine / alanine serine, also 16 masses change). The more distantly related cluster displayed both amino acid sub- stitutions (32 masses change) [3,4]. B. cereus strains also fall into two clades based on carbohydrate composition [5,7,8,19]. For comparison, B. anthracis vegetative cells (contains only galactose: clade 1 has an ‘‘intermediate’’ carbohy- drate profile (galactose and galactosamine), whereas clade 2 has a distinct profile (galactosamine)) and is distantly related. The car- bohydrate profiles and genotypic relatedness for the two clades have been recently shown to correlate [15]. A finer distinction using amplified fragment polymorphisms (AFLP) splits B. cereus into 10 groups with Branch F being the closest to B. anthracis. Hill et al. revealed that B. cereus and B. thuringiensis isolates are extensively interspersed with each other across all branches of the AFLP based phylogenetic tree. In contrast, B. * Corresponding author. Tel.: þ1 803 474 7777; fax: þ1 803 733 3192. E-mail address: ccallahan@gw.med.sc.edu (C. Callahan). Contents lists available at ScienceDirect Molecular and Cellular Probes journal homepage: www.elsevier.com/locate/ymcpr Molecular and Cellular Probes 22 (2008) 207–211 Contents lists available at ScienceDirect Molecular and Cellular Probes journal homepage: www.elsevier.com/locate/ymcpr 0890-8508/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.mcp.2007.11.003 Molecular and Cellular Probes 22 (2008) 207–211