Phylogenetic and protein prediction analysis reveals the taxonomically diverse distribution of virulence factors in the Bacillus cereus group

Bacillus cereus is a food contaminant with widely varying enterotoxic potential of its virulence proteins. In this article, phylogenetic analysis of the whole-genome amino acid sequences of 41 strains, evolutionary distance calculation of the amino acid sequences of the virulence genes, and functional and structural prediction of the virulence proteins were performed to reveal the taxonomically diverse distribution of virulence factors. The genome evolution of the strains showed a clustering trend based on the coding virulence genes. The strains of B. cereus have evolved into non-toxic risk and toxic risk clusters with medium-high- and medium-low-risk clusters. The distances of evolutionary transfer relative to housekeeping genes of incomplete virulence genes were greater than those of complete virulence genes, and the distance values of HblACD were higher than those of nheABC and CytK among the complete virulence genes. Cytoplasmic localization was impossible for all the virulence proteins, and NheB, NheC, Hbl-B, and Hbl-L1 were extracellular according to predictive analysis. Nhe and Hbl proteins except CytK had similar spatial structures. The predicted structures of Nhe and Hbl mainly showed ‘head’ and ‘tail’ domains. The ‘head’ of NheA and Hbl-B, including two α-helices separated by β-tongue strands, might play a special role in Nhe trimers and Hbl trimers, respectively. The ‘cap’ of CytK, which includes two ‘latches’ with many β-sheets, formed a β-barrel structure with pores, and a ‘rim’ balanced the structure. The evolution of B. cereus strains showed a clustering tendency based on the coding virulence genes, and the complete virulence-gene operon combination had higher relative genetic stability. The beta-tongue or latch associated with β-sheet folding might play an important role in the binding of virulence structures and pore-forming toxins in B. cereus.

distinct forms of food poisoning, the emetic type (uncommon) and the diarrheal type (common).

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To verify the above results and obtain more accurate molecular evolutionary relationships, we 165 established a second phylogenetic tree based on ANI analysis (Fig 2). According to enterotoxic risk

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The hblD and hblAD virulence proteins, which were observed in only two strains, were excluded. As

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shown in Table 1

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Examination of the phylogenetic tree constructed using the Hbl, Nhe, and CytK sequences (Fig 5) 218 showed that NheA and Hbl-L 2 as well as NheBC and Hbl-L 1 were more closely related to one another 219 than to the other components, and CytK was the least evolutionarily related. This result was also reflected 220 in the evaluation parameters of the 3-D enterotoxin protein structures. As shown in  in the pore, it is located at the edge of the β-sheet of the 'cap' region [36]. The 'rim' domain, which was

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In this study, forty-one strains of the B. cereus group were subjected to phylogenetic analyses based 273 on whole amino acid sequences. Enterotoxicity, which was evaluated on the basis of nheABC, hblACD,

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and cytK gene expression, was classified into levels of three types, two types, one type, and no types. In

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terms of evolutionary relationships, clusters of virulence and nonvirulence gene strains were evident, 16 276 and the regional distribution of the number of types of virulence genes was also presented, further 277 confirmed by ANI-based phylogenetic analyses. We found that the two phylogenetic trees were similar.

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All non-toxic-risk strains were concentrated in two clusters, and all but two of the medium-high-and higher relative genetic stability. The DV of hemolysin Bl was greater than that of nonhemolytic cytotoxin 291 K. nheABC, which was responsible for most of the cytotoxic activity of B. cereus isolates, showed stable, 292 strictly vertical inheritance [40]. In contrast to hbl, duplication or deletion of nhe, which was almost 293 exclusively transmitted vertically, was rarely observed, and cytK, a one-type gene, had the highest 294 relative genetic stability [12].

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Currently, it is commonly accepted that the toxicity potential of B. cereus is not driven by

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Cytotoxin K is a single protein with β-barrel pore-forming toxin in contrast to the tripartite toxin 331 complexes Hbl and Nhe. The CytK structure, which exhibits two 'latches' with many β-sheets folded 332 beside the 'cap' domain forming a β-barrel, was the pore structure on top of the conformation. The 'rim' 333 region, which was folded into a three-stranded antiparallel β-sheet, balanced the structure in the monomer.

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The predicted structure revealed that CytK was likely to belong to the leukocyte toxin family. These 335 monomers diffuse to target cells and are attached to them by specific receivers [54], which are lipids and 336 proteins that cause lysis of red blood cells by destroying their cell membrane [55].

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In this study, we describe the molecular evolution, function and structural diversity of virulence