General genome features.
The 5.31-Mb genome of
B. thuringiensis 97-27 comprises two replicons: a circular chromosome, encoding at least 5,198 open reading frames, and the pBT9727 plasmid (see Fig. S1A in the supplemental material). The 5.84-Mb genome of
B. cereus E33L comprises six replicons: a circular chromosome, encoding at least 5,682 open reading frames, and five plasmids (see Fig. S1B in the supplemental material).
B. thuringiensis 97-27 and
B. cereus E33L have broad similarities to and share a high degree of synteny with
B. anthracis Ames (
33),
B. cereus ATCC 14579 (
21), and
B. cereus ATCC 10987 (
32). Within the
B. cereus group,
B. anthracis,
B. cereus E33L, and
B. thuringiensis 97-27 are part of a distinct cluster which contains many pathogenic organisms (
18) (Fig.
1).
As illustrated in Fig. S2 in the supplemental material, a total of 3,917 putative proteins are shared among B. anthracis Ames, B. cereus ATCC 14579, B. thuringiensis 97-27, and B. cereus E33L using as a criterion whether genes were bidirectional best hits in BLAST searches. Comparison of the genomes of B. cereus E33L and B. thuringiensis 97-27 with B. anthracis Ames and B. cereus 14579 also identified strain-specific genes in each organism. Of the 5,682 predicted B. cereus E33L proteins, 253 of the chromosomally encoded genes and 416 of the plasmid genes are unique. Of the 5,197 predicted B. thuringiensis 97-27 proteins, 307 of the chromosomally encoded genes and 66 of the plasmid genes are unique. B. cereus E33L and B. anthracis Ames are the closest pair and share the highest number (221) of common proteins.
Virulence genes.
The chromosomally encoded virulence genes in
B. thuringiensis 97-27 and
B. cereus E33L are common to the
B. cereus group of bacteria (
14). Neither
B. thuringiensis 97-27 nor
B. cereus E33L has the highly characterized
B. anthracis toxin genes (
pag,
lef, and
cya) encoded on pX01 or the
cap genes encoded by pXO2 (
26,
27). Nonetheless, our results indicate that both
B. thuringiensis 97-27 and
B. cereus E33L share a set of virulence factors common to the members of the
B. cereus group. These common virulence genes include the three nonhemolytic enterotoxin genes (
nheABC), two channel-forming type III hemolysins, a perfringolysin O (listeriolysin O), a phosphatidyl-inositol-specific and a phosphatidyl-choline-preferring phospholipase, RNA polymerase sigma-B factor, and a p60 family extracellular protease. These last five genes are homologous to virulence genes encoded by the gram-positive pathogen
Listeria monocytogenes 12.
B. thuringiensis 97-27 and
B. cereus E33L also have a gene encoding cytotoxin K, which was previously identified in
B. cereus (ATCC 14579). While
B. cereus E33L lacks the
hbl operon, which is suspected as a primary factor in diarrheal
B. cereus food poisoning (
3),
B. thuringiensis 97-27 has the
hbl operon containing the hemolytic enterotoxin genes
hblCDBA also found in
B. cereus (ATCC 14579) (Fig.
2A). Interestingly, the
hbl gene cluster consists of the hemolytic enterotoxin (
hblCDBA) and other genes encoding the spore germination proteins
gerIABC, as well as other related proteins, that are ordered and oriented in a way that suggests their expression is coordinated by the transcriptional regulator TrrA. This gene cluster is part of a large, approximately 17.7-kb, 11-gene insertion (Fig.
2B). A degenerate IS
Rso11 transposase fragment is found at the presumed insertion boundary region, and direct repeats that overlap with the C-terminal UvrC-like protein were identified. This observation suggests a mechanism for the acquisition of these virulence factors in
B. thuringiensis 97-27,
B. cereus 14579, and
B. cereus G9241.
The opportunistic pathogenicity of
B. cereus and
B. thuringiensis may depend on the secretion of nonspecific extracellular virulence factors in response to transcriptional activation by PlcR (
34). However, in all
B. anthracis strains, the
plcR gene is inactivated by a frameshift mutation which creates an early stop codon (
1). In other
B. cereus isolates, the
plcR gene product up regulates the transcription of genes encoding enterotoxins, proteases, phospholipases, metabolic enzymes, proteins involved in motility and chemotaxis, proteins involved in sporulation, DNA metabolism, transcriptional regulators, and a variety of transporters by binding to a specific upstream motif (
1,
21,
34). The genes encoding PlcR appear intact in
B. thuringiensis 97-27 and in
B. cereus E33L. Analyzing the upstream sequences of coding regions for PlcR binding motifs identified genes likely to be activated by PlcR in
B. thuringiensis 97-27 and
B. cereus E33L. We found motifs upstream of most of the genes previously identified as potential members of a
plcR regulon in
B. cereus (
21) (see Table S1 in the supplemental material). Of particular interest are genes encoding probable virulence factors. In this respect, we found that the nonhemolytic enterotoxin genes (
nheA,
nheB, and
nheC) in both
B. thuringiensis 97-27 and
B. cereus E33L contained upstream PlcR motifs. In
B. thuringiensis 97-27,
B. cereus E33L, and
B. cereus (ATCC 14579), there are PlcR motifs upstream of cytotoxin K and several proteases, including collagenase, bacillolysin, enhancin, aminopeptidase Y, and peptidase T. In addition, we found PlcR motifs upstream of the phospholipase C and phosphatidylinositol-specific phospholipase C genes in all three genomes. Another gene that has an upstream PlcR motif in
B. thuringiensis 97-27,
B. cereus E33L, and
B. cereus (ATCC 14579) is error-prone DNA polymerase IV. This gene was previously suggested to induce adaptive point mutations that may affect pathogenicity. These observations (
21) support the hypothesis that differences in virulence among
B. anthracis,
B. cereus, and
B. thuringiensis are predominately due to alterations in gene expression rather than simple gain or loss of gene functions.
As
B. thuringiensis isolates usually contain
cry,
cyt, and/or
vip genes encoding insecticidal crystalline toxins, we compiled a list of 131
cry and
cyt gene sequences (
6), as well as the sequences of
vip3A (
11),
vip3V (
9),
vip1Ac and
vip2Ac (
38), and other more recently identified
cry gene sequences and blasted these sequences against the
B. thuringiensis 97-27 genome. There were no full-length hits to any of the query sequences. For the most part, the partial hits had low identities (under 40 to 50%) with the query sequences. Manual examination of the annotated
B. thuringiensis 97-27 genes that were partial hits and further analysis of these genes did not reveal any obvious candidates for
cry,
cyt, or
vip genes. Most of these
B. thuringiensis 97-27 genes had >80% amino acid sequence identity to other
B. cereus group (noninsecticidal) genes. So, we are confident that the genome of our current isolate contains no homologs of the known
cry,
cyt, or
vip genes. However, another possibility is that the plasmid encoding these genes was lost during culture.
Capsule biosynthetic genes.
Many microbial pathogens produce polymeric capsules that provide protection against host immune systems during the invasion process.
Bacillus species can produce both polysaccharide capsules that are common to many gram-positive and gram-negative species and the less common polyglutamic acid capsule. A summary of the capsule biosynthetic content in the sequenced members of the
B. cereus group is provided in the supplemental material (see Fig. S3). In
B. anthracis, three pXO2-encoded genes,
capB,
capC, and
capA, are required for synthesis of the polyglutamic acid capsule, and this structure plays a key role in the virulence of this organism (
12). To date, all
B. cereus group strains appear to contain a weak homolog of the pXO2
capA gene. This is also true in
B. cereus E33L, which contains a putative protein with 32% identity to
capA. However,
B. cereus E33L does not have a polyglutamic acid capsule (P. C. B. Turnbull, personal communication), nor does it appear to encode any genes involved in polysaccharide capsule synthesis (Table
1). Interestingly, the
B. thuringiensis 97-27 genome and
B. cereus 14579 encode a homolog of a member of a polysaccharide capsule synthesis pathway recently identified on a plasmid of the pathogenic
B. cereus G9241 (
19).
Sporulation and germination.
In anthrax, spores are the agent of infection. Spore formation occurs in response to nutrient limitation in the environment. In
B. subtilis, sporulation is initiated by a deficiency in carbon or nitrogen (
29) and is linked to changes in the expression of genes for degradative enzymes, such as alpha amylase, neutral protease, and alkaline protease (
20). The
B. subtilis spore coat is composed of at least 30 polypeptides, homologs of many of the
B. subtilis spore coat protein genes present in
B. anthracis (
23). We found differences in the number and composition of genes encoding spore coat proteins among
B. thuringiensis 97-27,
B. cereus E33L,
B. cereus ATCC 14579, and
B. anthracis (see Table S2 in the supplemental material). Both
B. anthracis and
B. cereus spores germinate in response to
l-alanine and ribosides. The germination response to
l-alanine and ribosides requires proteins of the
gerA family (
5).
B. thuringiensis 97-27,
B. cereus E33L,
B. cereus 569, and
B. cereus ATCC 14579 have a
gerI operon that is involved in an inosine-induced germination. The
gerI operon is homologous to the
gerA family operons of
B. subtilis (
5) and the
gerH operon in
B. anthracis (
41). Similarly, the
gerQ operon encodes germinant receptors that respond to inosine (
2).
B. thuringiensis 97-27 encodes
gerQ, while
B. cereus E33L does not.
Carbohydrate and amino acid utilization.
Like
B. cereus 14579 and
B. anthracis (
21,
33),
B. thuringiensis 97-27 and
B. cereus E33L appear predisposed to an environment rich in protein, having fewer genes for carbohydrate catabolism and more genes for amino acid metabolism (Table
2). For example, there are 12 carbohydrate polymer degradation genes in
B. anthracis Ames,
B. cereus 14579, and
B. thuringiensis 97-27 and 23 in
B. cereus E33L, compared to 41 in
B. subtilis. The
B. cereus group also appears to have reduced numbers of sugar-specific phosphoenolpyruvate-dependent phosphotransferase system genes. In contrast, members of the
B. cereus group have an expanded capacity for amino acid and peptide utilization. For example, there are 18 to 23 genes encoding peptide/amino acid ABC transporter-ATP binding proteins in the
B. cereus group, compared to 7 in
B. subtilis. There are six to nine genes for LysE family amino acid efflux system proteins in
B. cereus group members and only two in
B. subtilis. In addition to the expanded number of peptidase and protease genes in
B. cereus group species, 52 to 55 genes encode proteins involved in amino acid and amine catabolic pathways compared to 34 in
B. subtilis. These observations suggest that proteins, peptides, and amino acids may be a preferred nutrient source for all members of the
B. cereus group, which is consistent with the observations made previously (
21,
32).
Although the
B. cereus group species are closely related, variations in the sugar catabolism pathways are observed. Of particular note,
B. cereus E33L has 11 extra genes for carbohydrate polymer degradation compared with
B. anthracis Ames. Most of these are located on the large plasmid pE33L466. One of the most significant differences between
B. cereus E33L and other isolates in the
Bacillus cereus group is the large number of carbohydrate utilization gene clusters organized as operons on the pE33L466 plasmid (Fig.
3). These genes encode enzymes for myo-inositol degradation, galactose utilization, and pectin and gellan degradation. It is worth noting that the region of pE33L466 containing these four interlocked gene clusters is flanked by IS elements that were probably involved in their mobilization and integration into pE33L466. Figure
3 illustrates the metabolic pathways in which the products of these genes participate.
Antibiotic resistance.
The ecological niche and potential virulence of
B. thuringiensis 97-27 and
B. cereus E33L may be expanded through the presence of two lantibiotic resistance operons that are not present in
B. cereus or
B. anthracis. These include a mersacidin resistance operon consisting of
mrsR2,
mrsK2,
mrsF,
mrsG, and
mrsE and a salivaricin resistance operon consisting of
salY,
salK, and
salR. B. thuringiensis 97-27 and
B. cereus E33L have all of the genes in the mersacidin operon, while
B. anthracis strains A2012, Ames, and Sterne only have
mrsF. Although
B. thuringiensis 97-27 and
B. cereus E33L have all of the genes in the salivaricin and mersacidin resistance operons, they do not encode the
mrsA gene to produce mersacidin or the
salA gene to produce salivaricin. Therefore, these organisms can detect the presence of mersacidin and salivaricin produced by other bacteria but do not encode the capability to produce these lantibiotics themselves. Instead, the response may include increased expression of genes encoding other lantibiotics or virulence factors as previously suggested (
40).