Spo0B of Bacillus anthracis – a protein with pleiotropic functions

Spo0B of Bacillus anthracis – a protein with pleiotropicfunctionsAbid R. Mattoo, Mohd Saif Zaman, Gyanendra P. Dubey, Amit Arora, Azeet Narayan,Noor Jailkhani, Kusum Rathore, Souvik Maiti and Yogendra Singh

Allergy and Infectious Diseases, Institute of Genomics and Integrative Biology, Delhi, India

Bacillus anthracis is a Gram-positive, spore-forming

bacterium, and is the causative agent of anthrax.

Anthrax occurs when spores of B. anthracis gain access

to host tissues. In animals, this usually occurs by

ingestion, whereas in humans, spores usually enter the

host through inhalation or breaks in the skin barrier

[1]. The spores of B. anthracis are very resistant to

adverse environmental conditions. However, once

inside the host, spores are phagocytosed by macro-

phages or other innate immune cells, where they germi-

nate into vegetative cells. The lethality of the pathogen

is attributed to two major virulence factors: an anti-

phagocytic poly(d-glutamic acid) capsule and a toxin.

The anthrax toxin consists of three proteins: protective

antigen, lethal factor, and edema factor [2]. Hence,

spores are important agents in the spread of the dis-

ease. The process of spore formation in B. anthracis

remains a mystery.

Sporulation in Bacillus subtilis is regulated by the

phosphorelay signal transduction pathway, which is

Keywords

Bacillus anthracis; histidine kinase; Spo0B;

sporulation; Symbet

Correspondence

Y. Singh, Institute of Genomics and

Integrative Biology, Mall Road,

Delhi 110 007, India

Fax: 91 11 27667471

Tel: 91 11 27666156

E-mail: [email protected]

(Received 29 October 2007, revised 8

December 2007, accepted 12 December

2007)

doi:10.1111/j.1742-4658.2007.06240.x

Spo0B is an important component of the phosphorelay signal transduction

pathway, the pathway involved in the initiation of sporulation in Bacil-

lus subtilis. Bioinformatic, phylogenetic and biochemical studies showed

that Spo0B of Bacillus anthracis has evolved from citrate ⁄malate kinases.

During the course of evolution, Spo0B has retained the characteristic histi-

dine kinase boxes H, N, F, G1 and G2, and has acquired nucleotide-bind-

ing domains, Walker A and Walker B, of ATPases. Owing to the presence

of these domains, autophosphorylation and ATPase activity was observed

in Spo0B of B. anthracis. Mutational studies showed that among the six

histidine residues, His13 of the H-box is involved in the autophosphoryla-

tion activity of Spo0B, whereas Lys33 of the Walker A domain is associ-

ated with the ATPase activity of the protein. Thermodynamic and binding

studies of the binding of Mg-ATP to Spo0B using isothermal titration cal-

orimetry (ITC) suggested that the binding is driven by favorable entropy

changes and that the reaction is exothermic, with an apparent dissociation

constant (Kd) equal to 0.02 mm. The value of the dissociation constant

(Kd = 0.05 mm) determined by the intrinsic fluorescence of trytophan of

Spo0B was similar to that obtained by ITC studies. The purified Spo0B

of B. anthracis also showed nucleoside diphosphate kinase-like activity of

phosphate transfer from nucleoside triphosphate to nucleoside diphosphate.

This is the first evidence for Spo0B of B. anthracis as an enzyme with histi-

dine kinase and ATPase activities, which may have important roles to play

in sporulation and pathogenesis.

Abbreviations

GST, glutathione S-transferase; HATPase, histidine kinase ATPase domain; HK, histidine kinase; ITC, isothermal titration calorimetry;

NBD1, nucleotide-binding domain 1; NBD2, nucleotide-binding domain 2; Ndk, nucleoside diphosphate kinase; PknB, protein kinase B;

PVDF, poly(vinylidene difluoride); RR, response regulator.

FEBS Journal 275 (2008) 739–752 ª 2008 The Authors Journal compilation ª 2008 FEBS 739

activated by five histidine sensor kinases in response to

stress signals. The target of their activation is the

Spo0F response regulator (RR), to which they transfer

a phosphoryl group. The phosphoryl group is subse-

quently transferred to Spo0A through Spo0B, a phos-

photransferase [3]. This pathway is subjected to a

number of secondary controls that regulate the tran-

scription of various genes and the flow of phosphoryl

groups to Spo0A [4,5]. Once activated by phosphoryla-

tion, Spo0A promotes the transcription of a multitude

of genes required for sporulation, whereas it acts as a

repressor for certain genes expressed during the vegeta-

tive state [6]. Comparison of the protein sequences of

the phosphorelay components between B. subtilis and

B. anthracis revealed high homology in the phospho-

relay orthologs of Spo0F and Spo0A, whereas Spo0B

showed only 35% identity. Recently, nine genes have

been identified in the B. anthracis genome whose prod-

ucts may function as sporulation histidine sensor kin-

ases. Five of these sensor kinases were inferred to be

capable of inducing sporulation in B. anthracis,

whereas four of these were not associated with sporu-

lation. Sporulation kinase A (HisKA) of B. anthracis

(BA2636) is very similar to that of sporulation kina-

se A (KinA) of B. subtilis, but proof of its function in

sporulation remains elusive. Thus, unlike the situation

in B. subtilis, where KinA has a major role to play, the

initiation of sporulation in B. anthracis appears to be

regulated by the cumulative effect of more than one

kinase. Therefore, deletion of a single kinase does not

have any significant effect on sporulation of B. anthra-

cis [7]. It has also been shown that the virulence

plasmid-encoded sensor domains, pXO1-118 and

pXO2-61, of B. anthracis have a strong effect on the

activity of one of the nine sporulation sensor kinases,

BA2291. Expression of these sensor domains in

B. anthracis and B. subtilis has been shown to result in

the conversion of BA2291 from a sporulation kinase

to an enzyme that inhibits sporulation [8].

Comparative genomics studies have shown that the

genomic organization of various species of Bacillus is

not conserved, and it has been found that several genes

of B. cereus are more closely related to genes from other

bacteria and archea than to those from B. subtilis [9,10].

Moreover, some of the homologs of chromosomal genes

of B. subtilis have been found to be located on the

virulence plasmids, pX01 and pX02, of B. anthracis.

The genome of B. subtilis (4.2 Mb) is smaller than that

of B. anthracis (5.2 Mb), and comparison of the two

genomes shows that B. anthracis has some of the

important metabolic and developmental genes that are

absent in B. subtilis [11]. Comparison of the two-

component system of B. anthracis with the other

members of B. cereus group shows that B. anthracis

appears to lack some histidine kinases (HKs) and RRs,

and contains many truncated, possibly nonfunctional,

HK and RR genes. It has been hypothesized that

specialization of B. anthracis as a pathogen could have

reduced the range of environmental stimuli to which it

is exposed. This may have rendered some of its two-

component systems redundant, ultimately resulting in

the deletion of some HK and RR genes [12].

In B. subtilis, five sensor HKs feed phosphoryl

groups into the phosphorelay by recognizing a specific

signal and responding by catalyzing ATP-dependent

phosphorylation of a conserved histidine residue. Sub-

sequently, the phosphorelay system phosphorylates a

single domain response regulator, Spo0F, on a con-

served aspartate residue. The phosphoryl group is then

passed from the aspartate of Spo0F to a histidine side

chain on Spo0B [13,14]. The final step in the relay is

the transfer of phosphate to Spo0A at an aspartate

residue by phosphorylated Spo0B to activate its tran-

scriptional properties. Spo0B of B. subtilis has been

shown to be phosphorylated on His30, and mutation

in this residue abolished its phosphotransferase activ-

ity, resulting in the loss of sporulation [15]. It has been

shown that Spo0B forms a dimer, and each protomer

comprises two domains. One of the domains of Spo0B

mediates dimerization, forming a four-helical bundle

that closely resembles the phosphotransferase domains

of other sensor HKs [16]. Spo0B of B. anthracis is

quite different from that of B. subtilis, with only the

N-terminal domain showing similarity between the two

species. In this article, we show the evolutionary link

between Spo0B of B. anthracis and archetypal HKs. In

addition, some of the novel aspects of this protein that

may have important roles in the sporulation and vege-

tative growth of B. anthracis are also elucidated.

Results and Discussion

Phylogenetic analysis and diversity of Spo0B-like

sequences

A psi-blast search with Spo0B of B. anthracis against

a nonreduntant protein database revealed more than

100 sequences after five iterations (most of them were

Spo0B and citrate ⁄malate kinases). After five iterations

of psi-blast, no new significant sequences could be

retrieved. Among the retrieved sequences, those with

significantly high E values (> 10)15) were selected.

For this study, only those proteins were considered

that fulfilled two of the following criteria: SymBet,

synteny, conservation of CitA domain, and presence

of motif XRHD and lysine (characteristic of

Spo0B of Bacillus anthracis A. R. Mattoo et al.

740 FEBS Journal 275 (2008) 739–752 ª 2008 The Authors Journal compilation ª 2008 FEBS

GXXXXGKV of Spo0B). Of 100 sequences, only

33 fulfilled the criteria. We used the symmetrical ⁄bidirectional best hits (SymBet) method to define an

orthologous relationship between Spo0B and cit-

rate ⁄malate kinases ⁄hypothetical proteins. To resolve

cases where more than one SymBet was obtained, aux-

iliary criteria such as conservation of order of genes

(synteny) [17,18] and domain organization within the

protein were used to define orthologs. Synteny is par-

ticularly important in defining correct orthologs when

there are many highly similar sequences at different

genomic locations. The locus encompassing spo0B has

genes involved in ribosomal biosynthesis, protein syn-

thesis, development, sporulation and cell division; these

include obg (coding for GTP-binding protein), rpmA

(coding for ribosomal protein L27), prp (coding for

probable ribosomal protein), and rpIU (coding for

ribosomal protein L21) [19,20]. The synteny analysis

showed that some of the hypothetical proteins and cit-

rate ⁄malate kinases that were SymBet positive show

conserved genomic organization similar to spo0B of

B. anthracis and B. subtilis (supplementary Fig. S1A

and Table S1). The orthologs of Spo0B were found

to be present in some sporulating species of Clostrid-

ium, such as Clostridium tetani E88, Carboxydother-

mus hydrogenoformans Z-2901, Moorella thermoacetica

ATCC 39073, Pelotomaculum thermopropionicum SI,

Desulfitobacterium hafniense Y51, Syntrophomonas wol-

fei subsp. wolfei str. Goettingen (supplementary

Table S1). The hypothetical protein of Clostridium

novyi NT, which is SymBet negative, was also defined

as an ortholog of Spo0B on the basis of synteny. How-

ever, it has been reported that Spo0B is absent in

many sequenced genomes of Clostridium spp. such as

Clostridium botulinum and Clostridium difficile [21].

The ortholog of Spo0B in Thermoanaerobacter teng-

congensis, a nonsporulating species of order Clostridi-

ales that was SymBet positive (supplementary Fig. S1B

and Table S1) and showed identical synteny, was not

used for phylogenetic analysis, because it was trun-

cated. Another nonsporulating species of the Bacillales,

Exiguobacterium sibiricum 255-15, showed the presence

of an ortholog of Spo0B that was defined on the basis

of SymBet analysis.

Phylogenetic analysis of these sequences was per-

formed using full-length proteins (supplementary

Table S1). The phyletic analysis of protein sequences

revealed the segregation of the phenogram into clade I

and clade II (Fig. 1), with good bootstrap support val-

ues. Clade I included Spo0B of Bacillus spp., whereas

clade II included citrate ⁄malate kinases and some of

the true orthologs of Spo0B. The domain search

revealed the presence of CitA domains in majority of

these proteins. The domain organization became

more distinct in clade II, which also includes some of

the classic citrate ⁄malate kinases with distinct HK

ATPase (HATPase) domains (supplementary Table S1).

The Spo0B of B. anthracis present in clade I did not

show the presence of a CitA domain; however, this

domain was present in some of the members of

this clade (supplementary Table S1). The significant

bootstrap value (467) of the node and the presence

of a CitA domain in Spo0B and its orthologs in some

of the species of Bacillus and Clostridium suggest

a common origin of Spo0B from an ancestral

citrate ⁄malate-like kinase (Fig. 1, supplementary

Table S1).

Spo0B of B. anthracis was aligned with the HATP-

ase domain of CitA of Klebsiella pneumoniae and a

similar domain of DpiB of Escherichia coli, both of

which are classic and well-characterized citrate ⁄malate

kinases (Fig. 2A). The alignment suggested that Spo0B

of B. anthracis has a distinct H-box, N-box, and

F-box, whereas the G1-box and G2-box are distorted.

The alignment of Spo0B of B. subtilis with the HATP-

ase domains of the above two kinases under identical

parameters did not show the presence of these

domains. The protein sequence analysis of Spo0B of

B. anthracis showed the presence of a Walker A-like

domain (GXXXXGK). Therefore, further analysis was

carried out to compare the sequence of Spo0B with

ATPases. As a member of the Hsp100 ⁄Clp molecular

chaperone family, TClpB from Thermus thermophilus

has two nucleotide-binding domains, NBD1 and

NBD2, in a single polypeptide, each containing Walk-

er A and Walker B consensus motifs [22]. The multiple

sequence alignment of Spo0B of B. anthracis with the

two nucleotide-binding domains of TClpB showed the

presence of Walker A and Walker B motifs with con-

sensus sequences GXXXXGKV and xxxxDE respec-

tively (Fig. 2B). Interestingly, B. subtilis Spo0B did not

show the presence of these domains under the same set

of conditions used for multiple sequence alignment

(data not shown). The multiple sequence alignment of

the Bacillus species showing the distribution of the HK

and ATPase motifs is represented in supplementary

Fig. S2. The alignment indicated that the important

motifs required for ATP binding in B. anthracis and

other Bacillus cereus family members are distorted in

B. subtilis. These results suggest that, in addition to

the presence of HK domains, Spo0B of B. anthracis

has acquired Walker A and Walker B domains during

evolution. Thus, Spo0B represents an interesting snap-

shot of the evolution of noncanonical HKs and may

have important roles in sporulation and other func-

tions in B. anthracis.

A. R. Mattoo et al. Spo0B of Bacillus anthracis


Dimerization of Spo0B

Autophosphorylation in most HKs occurs by a trans-

phosphorylation reaction that requires dimer forma-

tion [16]. Spo0B of B. subtilis has been reported to be

a dimer, each monomer consisting of two domains

[14]. It has been reported that in ATPases and some

members of the gyrase–Hsp90–HK–MutL family, ATP

binding induces intersubunit contacts between the

nucleotide-binding domains in the homodimer or

higher oligomers [24,25]. The dimerization of the

nucleotide-binding domains may be critical for ATP

hydrolysis. Spo0B of B. anthracis is different from

Spo0B of B. subtilis, as only the N-terminal domain is

conserved between the two, with an overall identity

of 35%. Spo0B of B. anthracis shows the presence of

both HK and ATPase domains. To elucidate whether

Spo0B of B. anthracis can dimerize, crosslinking was

done using glutaraldehyde, as described in Experimen-

tal procedures. Spo0B of B. anthracis showed the

phenomenon of dimerization (Fig. 3).

Autophosphorylation and nature of the

phosphorylated amino acid in Spo0B

The in silico studies indicated the link between Spo0B

and citrate ⁄malate kinases and ATPases. The N-termi-

nal domain of Spo0B of B. subtilis closely resembles

Clade I

Clade II

10 0

gi|6805517 E. sibiricum 255-15

gi|5696330 B. clausii KSM-K16

gi|8909898 Bacillus sp. NRRL B-14911

gi|5642114 G. kaustophilus HTA426

gi|8920028 B. cereus cytotoxis 391-98

gi|8920524 B. wehenstephanensis

gi|4756663 B. cereus G9241

gi|3002251 B. cereus ATCC 14579

gi|7576316 B. thuringenesis525

374

gi|3026451 B. anthracis

gi|5214106 B. cereus E33L563

595

89 8

99 6

74 8

23 2

gi|1561377 B. halodurans C-125

gi|1607984 B. subtilis

gi|5208127 B. licheniformis ATCC 1458099 9

50 0

688

gi|2309949 O. iheyensis HTE831

gi|1145671 S. wolfei

gi|7804322 C. hydrogenoformans

gi|2821167 C. tetani E88

gi|1184447 C. novyi NT961

35 4

gi|8989591 D. hafniense Y51

gi|1096492 D. hafniense DCB-2996

228

gi|8358942 M. thermoacetica ATCC 39073

gi|9866161 P. thermopropionicum SI

gi|8894492 D. reducens MI-1916

gi|9498494 D. geothermalis

gi|5597835 T. thermophilus547

gi|1705888 K. pneumoniae

gi|1096481 D. hafniensie DCB-255 7

69 1

gi|8989527 D. hafniense Y51


gi|8358975 M. thermoacetica ATCC 3907341 8

392

242


gi|8250116 C. saccharolyticus DSM 890337 3

357

29 0

17 2

32 5

794

57 5

46 7

Fig. 1. Phylogenetic analysis of Spo0B of Bacillus anthracis. Analysis was performed by the neighbor-joining method of PHYLIP (v. 3.63), and

the tree was viewed with TREEVIEW. The number at the nodes indicates bootstrap support values after 1000 bootstrap cycles. The unrooted

tree was drawn using hypothetical protein gi|68055173 of Ex. sibiricum 255-15 as outgroup. The tree was divided into two clades according

to criteria discussed in Results and Discussion. E., Exiguobacterium; B., Bacillus; C. hydrogenoformans, Carboxydothermus hydrogenofor-

mans; C., Clostridium; M., Moorella; K., Klebsiella; D. hafniense, Desulfitobacterium hafniense; D. geothermalis, Deinococcus geothermalis;

T., Thermus; C. saccharolyticus, Caldicellulosiruptor saccharolyticus; S. wolfei, Syntrophomonas wolfei; O., Oceanobacillus; G., Geobacillus;

D. reducens, Desulfitobacterium reducens; P. thermopropionicum, Pelotomaculum thermopropionicum.



the dimerization histidine phosphotransfer domain of

HKs, and its C-terminal domain is topologically simi-

lar to the catalytic ATP-binding domain of EnvZ and

CheA. The major difference in the C-terminal domain

of Spo0B of B. subtilis is that it has a Bergerat ATP-

binding fold but lacks the conserved residues for ATP

binding [26,27]. It has been shown that nucleoside

diphosphate kinase (Ndk), an HK, is autophosphory-

lated even in the absence of ATP-binding motifs [28].

The sequence comparison of Spo0B with the HATPase

domain of CitA of K. pneumoniae and the similar

domain of DpiB of E. coli, suggested that Spo0B of

B. anthracis was different from Spo0B of B. subtilis.

The autophosphorylation activity of Spo0B of B. an-

thracis was evaluated by incubating purified protein

with [32P]ATP[cP] at 37 �C for 30 min. Proteins were

separated by 12% SDS ⁄PAGE and analyzed by auto-

radiography. A sharp band at the expected size of

24.6 kDa was observed, indicating that Spo0B is an

autophosphorylating enzyme (Fig. 4A). To confirm the

autophosphorylation activity, glutathione S-transferase

(GST)-tagged Spo0B was also subjected to autophos-

phorylation, and showed a band on the autoradiogram

at the expected size of 50 kDa (data not shown). To

further confirm that the autophosphorylation was not

due to contaminating proteins, 1 mm ATP and

A

B

Fig. 2. (A) Sequence alignment of Bacillus anthracis Spo0B (gi|30264510) with the HATPase domains of CitA of K. pneumoniae (gi|1705888)

and DpiB of E. coli (gi|26246600). The CLUSTAL W program was used for the alignment under the following conditions: ktup = 2, matrix = id,

gap open = 2, gap extension = 0.5. Alignments were further manually edited. Identical residues in all three sequences are indicated with

astrerisks; colons denote conserved substitutions. H, X, N, G1, F and G2 are shown in boxes. (B) Sequence alignment of B. anthracis

Spo0B with NBD1 and NBD2 of molecular chaperone TClpB. The alignment was done using the T-COFFEE program. The Walker A and

Walker B domains are represented as GXXXGKT and xxxxDE.



5 mm MgSO4 were added to the lysis buffer and incu-

bated at 37 �C for 10 min. It is known that the ATP

and MgSO4 mixture releases the contaminating pro-

teins and chaperones from the proteins [29]; Spo0B

retained its autophosphorylation activity in the pres-

ence of this mixture, indicating that this activity is

intrinsic to the protein.

To determine the nature of the phosphorylated

amino acid, autophosphorylated proteins were incu-

bated in acid or base as described in Experimental

procedures. Phosphorylated histidines form phospho-

ramidate bonds that are sensitive to acid and resistant

to base, whereas phosphorylated serine and threonine

produce phosphoester bonds that are acid resistant

and base labile. Moreover, tyrosine phosphorylation is

resistant to both acid and base, whereas aspartate

phosphorylation is labile in both acid and base [30].

The phosphorylated Spo0B showed acid sensitivity,

indicating phosphoramidate bond formation as

reported for HKs (Fig. 4B). In this experiment, protein

kinase B (PknB) and Ndk of Mycobacterium tuberculo-

sis were used as controls [31–33].

The HKs autophosphorylate at a histidine; the phos-

phoryl group from histidine is transferred to a con-

served aspartate of the RR. The catalytic mechanism is

well understood for the aspartyl phosphorylation,

whereas the autokinase reaction is not well under-

stood. This is due to the smaller amount of structural

information available for the HKs. Mutational analysis

of the important residues of the N-box, G1-box, F-box

and G2-box of the catalytic domain of HKs has shown

that individual mutations of these residues do not

result in kinase-dead forms of these kinases, although,

in a few cases, these mutations reduced ATP binding

1 2 3

Coomassie staining

1 2 3 4

Autoradiogram

PknB mNdk Spo0B

NaOHB

A C

HCL

PknB mNdk Spo0B

Fig. 4. (A) Autophosphorylation of Spo0B. Autophosphorylation of Spo0B was measured by incubation of purified protein (2 lg) with

[32P]ATP[cP] in the presence of varying amounts of MgCl2 (lanes 1–3: 1, 2 and 5 mM MgCl2, respectively), followed by separation by

12% SDS ⁄ PAGE. The autoradiogram was developed on a phosphorimager. (B) Acid–base stability of phosphorylated Spo0B. The phosphory-

lated proteins were transferred to a PVDF membrane and treated with 6 M HCl or 1 M NaOH, as described in Experimental procedures.

Spo0B is acid labile and base stable, as observed for mNdk, an HK of M. tuberculosis. (C) Autophosphorylation of Spo0B mutants. Spo0B

(lane 1) and mutants H13A (lane 2), H16A (lane 3) and H37A (lane 4) were incubated with [32P]ATP[cP], and this was followed by separation

by 12% SDS ⁄ PAGE. The gel was developed in a phosphorimager and then stained with Coomassie Briliant Blue. Mutation of His13 (H13A)

caused a loss of autophosphorylation activity.

25 °C 37 °C

21

31

45

66

0 0.01 0.05 0.1 0 0.01 0.05 0.1M

Monomer

Dimer

% Glutaraldehyde

Fig. 3. Dimerization of Spo0B. His6-tagged Spo0B (2 lg) was incu-

bated with increasing concentrations of glutaraldehyde at two dif-

ferent temperatures (25 �C and 37 �C). The samples were resolved

by 10% SDS ⁄ PAGE, and the gel was stained with Coomassie Bril-

liant Blue. The figure illustrates the ability of Spo0B to dimerize,

which is an important property of HKs and ATPases.



and autophosphorylation. ATP binding and its utiliza-

tion is a cumulative effect of these motifs [34,35].

However, mutations that alter the conserved histidine

of the H-box consistently disrupt the autophosphoryla-

tion activity of HKs [36,37]. This is in contrast to what

is seen for the serine ⁄ threonine kinases, where muta-

tion of the catalytic lysine crucial for ATP binding

results in complete loss of kinase activity [38–40]. Stud-

ies with serine ⁄ threonine kinases have suggested that

autophosphorylation occurs on more than one residue

[41]. In addition, serine ⁄ threonine kinases are known

to phosphorylate exogenous substrates such as histone

and myelin basic proteins, whereas no such substrates

are known for HKs [38,40].

The acid–base test showed that Spo0B behaves as

an HK. Immunoblot analysis of Spo0B with mono-

clonal anti-phosphoserine, anti-phosphothreonine and

anti-phosphotyrosine showed that this protein does

not belong to the serine ⁄ threonine ⁄ tyrosine group of

kinases (data not shown). Spo0B has six histidine resi-

dues, and efforts were made to map the histidine resi-

due involved in the autophosphorylation reaction by

mutagenesis studies. His13 and His16 of the H-box are

conserved among the Bacillus species (supplementary

Fig. S2). The two residues of the H-box were mutage-

nized; mutation of His13 resulted in reduced autophos-

phorylation, whereas mutation of His16 had no effect

on the activity of Spo0B (Fig. 4C). Mutation of the

other histidine residues of Spo0B, such as H37A

(Fig. 4C), H87A, H91A, and H109A (supplementary

Fig. S3), had no effect on the autophosphorylation

activity of Spo0B. It has been reported that nine spor-

ulation kinase genes are present in B. anthracis [7].

Two of these showed frameshifts in all B. anthracis

strains, whereas one of them was inactivated in the

pathogenic strain of B. cereus, harboring the B. anthra-

cis toxin plasmid pX01. These facts suggest that, evo-

lutionarily, B. anthracis differs significantly from the

other species of the genus Bacillus, and the presence of

the virulence plasmids may have rendered some of the

important sensor kinases of B. anthracis nonfunctional.

Thus, the possibility exists that it may have acquired

dissimilar machinery from that of B. subtilis for the

initiation of sporulation.

ATPase activity of Spo0B

Bioinformatic analysis suggested the presence of Walk-

er A and Walker B domains in Spo0B, a characteristic

of ATPases. Experiments with Spo0B showed the pres-

ence of ATPase activity, as evidenced by the decrease

in the amount of [32P]ATP[cP] and the simultaneous

increase in the amount of 32Pi (Fig. 5A). Earlier

reports on ATPases suggested that the lysine in the

Walker A motif directly interacts with the phosphate

group of bound ATP (GXXXXGK). The acidic resi-

dues within the Walker B motif play an essential role

ATP

Pi

A

B

1 2 3 4 5 6 7

02000400060008000

10 00012 00014 00016 00018 00020 000

0 15 30 60

Time (min)

Inta

ct A

TP

(im

ager

un

its)

H16AH38AK33AK33RSpo0B

Fig. 5. (A) ATPase activity of purified Spo0B. Purified Spo0B (2 ug)

was incubated with 10 lCi of [32P]ATP[cP] at 37 �C for various time

periods (0–90 min), and hydrolysis of ATP was monitored as an

indicator of ATPase activity. Lane 1: [32P]ATP[cP] alone. Lanes

2–6: [32P]ATP[cP] with Spo0B at 5, 15, 30, 45 and 60 min, respec-

tively. Lane 7: [32P]ATP[cP] with Spo0B + 10 lM EDTA. The addi-

tion of EDTA resulted in the loss of ATPase activity, which shows

that MgCl2 is essential for this activity. (B) ATPase activity of

mutants of Spo0B. Purified Spo0B (2 lg) and equal amounts of its

mutants H16A, His38A, K33A and K33R were incubated with

10 lCi of [32P]ATP[cP] at 37 �C for various time periods (0–60 min)

and separated by poly(ethylenimine) TLC. The signal intensity of

remaining ATP was measured on a phosphorimager (defined as

imager units) to determine the ATPase activity of mutants. Each

value is an average ± SE of three individual experiments. Mutations

in the Walker A motif (K33A, K33R) reduced the ATPase activity of

Spo0B; however, mutation in an adjacent residue (H38A) had no

effect on ATPase activity.



in coordinating the Mg2+ and probably the water

molecule that attacks the b–c bond of the ATP. Con-

sistent with this hypothesis, it has been postulated that

the Walker B motif is involved in ATP hydrolysis

rather than ATP binding [42,43]. Mg2+ is important

for ATP binding, and without this divalent cation,

ATP does not bind well. The mutation of lysine, which

is involved in binding of ATP, has been shown to

abolish the ATPase activity [44,45]. Conservative sub-

stitution of the lysine in the Walker A motif is pre-

dicted to result in a protein capable of limited binding,

but not hydrolysis, of ATP [46]. These observations

were tested with Spo0B of B. anthracis. The mutation

of lysine to alanine (K33A) or arginine (K33R) in

Spo0B resulted in the loss of the ATPase activity of

Spo0B (Fig. 5B). Mutation of His38 (H38A), which lies

in the vicinity of the Walker A domain, had no effect on

the ATPase activity of Spo0B. Similar results were

obtained by mutating His16 (H16A) of Spo0B. As

Spo0B is an enzyme with both HK and ATPase motifs,

the ATPase activity is not abolished completely by

mutation of the lysine of the Walker A domain, as

reported for other ATPases. The residual ATPase

activity could be due to the presence of the HK motifs.

Ndk-like activity in Spo0B

The purified Spo0B of B. anthracis showed Ndk-like

phosphotransferase activity, as it transferred the termi-

nal phosphate from [32P]ATP[cP] to all nucleoside

diphosphates (Fig. 6), converting them into the corre-

sponding triphosphates [28]. The phosphotransferase

activity of Spo0B with UDP and CDP as substrates

was similar to that observed with Ndk of M. tuberculo-

sis, whereas, with GDP as a substrate, the activity was

quite low (Fig. 6). NdK is an important cellular

enzyme that monitors and maintains nucleotide pools

and has been implicated in a number of regulatory

processes, including signal transduction and develop-

ment [33]. Moreover, Ndk-like activity has been

observed in a number of proteins that are not classic

Ndk enzymes [23,47–50]. It has been shown that tran-

scription of the spo0B gene in B. subtilis occurs during

vegetative growth and that expression decreases during

sporulation [51]. The intracellular levels of GTP or

UTP and ATP have been shown to affect the level of

sporulation in B. subtilis [52,53]. The presence of

Spo0B in a few nonsporulating species of the Bacill-

ales, such as Ex. sibiricum 255-15, suggests that, in

addition to sporulation, Spo0B may have other roles

to play, such as maintaining the nucleotide pool.

ATP binding to Spo0B

The thermodynamics of binding of ATP to Spo0B was

investigated by isothermal titration calorimetry (ITC)

[54–56]. This method directly measures the heat of

reaction (enthalpy, DH), stoichiometry of substrate

binding (n) and the dissociation constant of the sub-

strate (Kd) required to determine the Gibbs free energy

of association (DG = )RT ln 1ÆKd)1) and entropy

(TDS = DH)DG). The typical titrations of Spo0B

with the nucleotide substrate Mg-ATP is shown in

Fig. 7Aa. The titrations of Mg-ATP at 100 mm of

Spo0B yielded a Kd of 0.02 mm.

The thermodynamic parameters of binding are sum-

marized in Table 1. The binding of Mg-ATP yielded

DH and )TDS values of ()3341 ± 25) calÆmol)1 and

(3069.4 ± 45) calÆmol)1 respectively, which shows that

binding of nucleotide to Spo0B is driven by favorable

entropy changes and that the reaction is exothermic.

Data analysis gave a best fit to a single-site binding

model with a stoichiometry of 0.83 mol per mole of

Spo0B (Fig. 7Aa). ITC studies of ATP binding to the

K33A mutant of Spo0B showed a considerable

decrease in the ATP binding, which further supports

the idea that Lys33 of the Walker A domain is

required for ATP binding (Fig. 7Ab). Studies of the

binding of ATP to H13A showed similar binding and

energy changes as observed in Spo0B (data not

shown). The ITC results were confirmed by measuring

changes in the intrinsic tryptophan fluorescence [57,58]

of Spo0B upon addition of ATP. Trp18 in the H-box

and Trp149 in the distorted G2-box are the two trypto-

phans that are important for measurement of the

ATP

Ndk+

CDP

Spo0B

+CDP Ndk

+UDP

Spo0B+U

DP

Spo0B

+GDP Ndk+

GDP

GTP

CTP

UTP

Fig. 6. Nucleoside diphosphate kinase activity of Spo0B. Purified

Spo0B or mNdk (2 lg) were incubated with 10 lCi of [32P]ATP[cP]

and 1 mM NDP (CDP or UDP or GDP) for 30 min at 37 �C. Reac-

tions were stopped by addition of 5 lL of 5 · SDS loading dye and

resolved by poly(ethylenimine) TLC.



intrinsic tryptophan fluorescence of Spo0B (Fig. 7Ba).

Titration of Spo0B by ATP in the presence of magne-

sium ions decreased the fluorescence intensity by

more than 50% before reaching saturation (Fig. 7Bb).

The decrease in the intrinsic fluorescence at 340 nm

was monitored by increasing the concentration of

ATP, which allowed determination of the Kd value

(Kd = 0.5 mm). The Kd value obtained by fluorescence

measurement was similar to the value obtained by

ITC. The binding of ATP enabled us to elucidate the

nucleotide-binding characteristics of Spo0B in more

detail, showing that Spo0B has the necessary motifs

for binding of ATP, and thus further confirms that

Spo0B of B. anthracis has the propensity to autophos-

phorylate and that it has ATPase activity.

In conclusion, we show that Spo0B of B. anthracis

has acquired many new features and shows multiple

activities, in contrast to its corresponding ortholog in

B. subtilis, which acts only as a phosphotransferase. It

seems that this protein has been under tremendous

evolutionary pressure, because of which it may not

conform to the conventional definition of an ortholog.

The pathogenicity of B. anthracis may be one of the

important reasons for this change, and the unique fea-

tures of Spo0B may have important roles to play in

the sporulation and survival of the pathogen.

A a b B

a

b

Fig. 7. (A) Binding of ATP to Spo0B using the ITC method. Equal amounts of protein solution (100 lM) of Spo0B (a) and K33A (b) were

titrated with the ATP (730 lM) solution in the buffer containing MgCl2 at 25 �C. The data were fitted to a model for a single class of binding

sites (solid line). ORIGIN 7.0 software was used to fit the thermodynamic parameters to the heat profiles. The results indicate that Spo0B has

the necessary motifs for binding of ATP, and mutation of lysine (K33A) affects the binding of ATP to Spo0B. (B) Characterization of Spo0B–

ATP interaction by trytophan intrinsic fluorescence. (a) Location of Trp18 and Trp149 in the vicinity of the ATP-binding sites of Spo0B. Trp18

is located between the H-box and Walker A domain, whereas Trp149 is present in the distorted G2-box. H, N, G1, F and G2 represent HK

motifs, and WA and WB represent the Walker A and Walker B domains. (b) Fluorescence spectra of Spo0B in the presence of increasing

concentrations of ATP (375 nM to 75 lM). All spectra were corrected by subtraction of spectra obtained in buffer alone and buffer + ligand.

The dissociation constant Kd for the Spo0B–ATP complex was determined from the hyperbolic plot shown in the inset. These data further

confirm the ATP-binding ability of Spo0B as observed in ITC experiments.

Table 1. Thermodynamic parameters of nucleotide binding to Spo0B. The values are obtained by fitting the ITC titration data by applying the

single-site model. The values of DG were calculated from the association constants (Ka). The values are means ± SE of three individual mea-

surements. T is the temperature in kelvins (1 cal = 4.186 J).

Nucleotide Ka (· 10)5M

)1) Kd (mM) DH (calÆmol)1) )TDS (calÆmol)1) DG (calÆmol)1)

Mg-ATP 0.49 0.02 )3341 ± 25 3069.4 ± 45 )6401.21



Experimental procedures

Chemicals and bacterial strains

The B. anthracis Sterne strain was used for the isolation

of genomic DNA. E. coli strains DH5-a and BL21-DE3

were used for plasmid transformation. Biochemicals,

reagents and chromatography materials were purchased

from Sigma-Aldrich (St Louis, MO, USA), Merck

(Darmstadt, Germany) and Bangalore Genie India Ltd.

(Bangalore, India). Bacterial culture media were pur-

chased from HiMedia laboratories (Mumbai, India). Res-

ins for affinity purification Ni–nitrilotriacetic acid and

GST–Sepharose were purchased from Qiagen (Hilden,

Germany) and Amersham Biosciences (Uppsala, Sweden).

DNA-modifying enzymes were obtained from Roche

(Basel, Switzerland). Radiolabeled [32P]ATP[cP] and

[32P]GTP[cP] were purchased from BRIT (Hyderabad,

India).

Bioinformatic and phylogenetic analysis

Spo0B of B. anthracis was used as a query sequence to

retrieve homologous sequences from a nonredundant pro-

tein database using psi-blast [59] with default settings.

Multiple sequence alignments were constructed using

clustalx (1.83) [60] or t-coffee (v. 4.59). For phyloge-

netic analysis, the sequences were initially aligned using the

program t-coffee, v. 4.59 [61]. A distance matrix of pair-

wise comparisons of the proportion of different amino acids

per site was constructed using the program protdist of

phylip v. 3.572c. The programs seqboot, neighbor and

consense [62] were used to derive a neighbor-joining tree

that was replicated in 1000 bootstraps. The Jones–Taylor–

Thomton amino acid substitution matrix was used in prot-

dist. The input order of sequences for phylogenetic analysis

was randomized, wherever this option was given. The phy-

logenetic tree was visualized with treeview. Domain

boundaries and structural organization of the retrieved

Spo0B sequences were analyzed using smart [63], inter-

proscan (http://www.ebi.ac.uk/InterProScan/), and con-

served domain Database (http://www.ncbi.nlm.nih.gov/

Structure/cdd/wrpsb.cgi).

Plasmid construction and mutagenesis

Plasmid construction and mutagenesis were done as

described earlier [40]. The B. anthracis Sterne strain was

used as a template for PCR-based amplification of the

BAS4338 gene coding for Spo0B (length of gene, 549 bp).

The nucleotide sequences of the two primers were FP-1 (5¢-GCC ATG GGG ATC CCC ATG AAT AAA AAA TGG

ACA C-3¢), carrying a BamHI site at the 5¢-end (forward

primer), and RP-1 (5¢-CGT AGG CCT TTG AAT TCC

TTA TTT CAC CAC ACT G-3¢), carrying an EcoRI site

(reverse primer). The amplified product was digested with

BamHI and EcoR1, and the resulting fragment was inserted

into the pROEX-HTc plasmid, which was previously

digested with the same restriction enzymes. The vector

pROEX-HTc has sequences coding for six histidine residues

at the N-terminus. The recombinant plasmid was desig-

nated pSpo0B. Spo0B cloned in pPROEX-HTc was

digested with BamH1–Xho1 and subcloned into the same

sites of pGEX-5X3 (Amersham Biosciences), which has

sequences coding for GST at the N-terminus. Site-directed

mutagenesis of His13, His16, His38, His87, His91, His109

and lysine was performed by using primers carrying the

desired changes (supplementary Table S2). All the experi-

ments were performed with Spo0B containing six histidine

residues at the N-terminus unless otherwise specified.

Purification of Spo0B and its mutants

The Spo0B and the mutant proteins were purified as

described previously [40]. In brief, E. coli BL21-DE3 was

transformed with recombinant plasmid pSpo0B. E. coli

carrying recombinant plasmid was grown in Luria broth

containing 100 lg of ampicillin per mL at 37 �C with

shaking at 250 r.p.m. When D600 nm reached 0.6, isopropyl

thio-b-d-galactoside was added to a final concentration

of 1 mm. After 3 h of induction, the cells were pelleted in a

centrifuge at 5000 g for 10 min. For purification of protein,

200 mL of culture pellet was suspended in 5 mL of sonica-

tion buffer (50 mm Tris, pH 8.5, 5 mm b-mercaptoethanol,

300 mm KCl, 1 mm phenylmethylsulfonyl fluoride). Cells

were sonicated at 4 �C (30 s burst, 30 s of cooling, 6 W

power) for eight cycles. The cell lysate was centrifuged

at 15 000 g for 30 min. The supernatant was mixed with

2 mL of Ni–nitrilotriacetic acid resin equilibrated previ-

ously with sonication buffer. The slurry was packed into a

column and allowed to settle. The matrix was washed first

with sonication buffer, and then with wash buffer

(20 mm Tris, pH 8.5, 1 m KCl, 10% glycerol, 5 mm b-mer-

captoethanol). Protein was eluted with a linear gradient

of 0 and 500 mm imidazole in elution buffer (20 mm Tris,

pH 8.5, 100 mm KCl, 10% glycerol). Fractions of 1 mL

were collected and analyzed by 12% SDS ⁄PAGE. The

fractions containing purified Spo0B were stored at )70 �C.The protein was dialyzed to remove imidazole, using buffer

(20 mm Tris, pH 8.5, 200 mm KCl), before being used for

the biochemical assays.

Dimerization assay of Spo0B

The ability of Spo0B to form dimers was assessed by chem-

ical crosslinking using glutaraldehyde, as described previ-

ously [64]. Purified Spo0B was dialyzed against crosslinking

buffer (20 mm Tris, pH 7.4, 5 mm MgCl2, 100 mm KCl,

1 mm b-mercaptoethanol, 5% glycerol), and 2 lg of Spo0B

was then incubated with different concentrations of glutar-



aldehyde (0.01%, 0.05%, and 0.1%) at 25 �C at 37 �C for

30 min. The reactions were stopped by the addition of

5 · SDS loading dye, subjected to 10% SDS ⁄PAGE, and

visualized by Coomassie Brilliant Blue staining.

Autophosphorylation of Spo0B and mutants

Autophosphorylation activity of the purified Spo0B and

mutant proteins was checked as described previously [28].

In brief, 2 lg of the purified Spo0B or mutant proteins

was incubated with 10 lCi of [32P]ATP[cP] in a final

reaction volume of 20 lL prepared with TMD buffer

(20 mm Tris ⁄HCl, 5 mm MgCl2, 1 mm dithiothreitol,

pH 7.4). The reaction was allowed to continue for 30 min

and terminated by addition of 2 lL of 5 · SDS sample buf-

fer. The samples were boiled for 10 min and separated by

12% SDS ⁄PAGE. The gel was fixed in 40% methanol,

dried, and evaluated in an FLA 2000 (Fujifilm) phosphor-

imager after exposure for 30 min.

Acid–base stability assay

Autophosphorylation reactions were performed as

described above, using equal amounts (2 lg) of Spo0B

(B. anthracis), Ndk and PknB (M. tuberculosis) in duplicate.

The 32P-labeled proteins were separated on polyacrylamide

gel, and the proteins were transferred to poly(vinylidene

difluoride) (PVDF) membranes. The transfer of labeled

proteins was checked on a phosphorimager. One PVDF

membrane was incubated at 65 �C for 2 h in 6 m HCl and

the second was incubated at 65 �C for 2 h in 1 m NaOH.

After the treatment, the PVDF membrane was again

checked in the phosphorimager [30].

ATPase activity of Spo0B

ATPase activity was measured by incubating Spo0B (2 lg)with 10 lCi of [32P]ATP[cP] in a buffer consisting of

20 mm Tris ⁄HCl (pH 7.4), 1 mm MgCl2, 1 mm dithiothrei-

tol and 1 mgÆmL)1 BSA at 37 �C [28]. Five microliter vol-

umes of samples were removed at different time intervals.

The reaction was stopped by addition of 1 lL of 5 · SDS

sample buffer. A reaction mixture of 2 lL from each time

interval was loaded onto the polyethyleneimine cellulose F

TLC plate, and resolved using 0.75 m KH2PO4 (pH 3.75)

as the moving phase. The TLC plate was dried and exposed

in a phosphorimager.

Ndk-like activity of Spo0B

The Ndk-like activity of purified Spo0B was assayed as

described previously [28]. In brief, 2 lg of purified protein

was incubated with 1 mm (final concentration) each NDP

(where N is G, C or U) and 10 lCi of [32P]ATP[cP] in a

final volume of 20 lL of TMD buffer. The reaction was

initiated by the addition of ATP, and continued for 10 min

at room temperature. Then, 2 lL of 5 · SDS sample buffer

was added to stop the reaction. The 2 lL reaction mixture

was spotted on a polyethyleneimine cellulose F TLC plate,

resolved using 0.75 m KH2PO4 (pH 3.75) as the moving

phase, and visualized by autoradiography.

ITC experiments

ITC was performed using a MicroCal VP-ITC-type micro

calorimeter (MicroCal Inc., Northampton, MA, USA)

at 25 �C [54–56]. Temperature equilibration prior to experi-

ments was allowed for 1–2 h. All solutions were thoroughly

degassed before use by stirring under vacuum. Protein and

titrating ligand samples were prepared in the same dialysis

buffer (20 mm Tris, pH 7.4, 5 mm MgCl2). The pH of

nucleotide solutions was carefully checked, and if necessary,

adjusted to pH 7.4. A typical titration experiment consisted

of consecutive injections of 10 lL of the titrating ligand (in

approximately 40 steps, at 5 min intervals, into the protein

solution in the cell with a volume of 2 mL). The titration

data were corrected for the small heat changes observed in

the control titrations of ligands into the buffer. Data analy-

sis was performed with origin 7.0 software, provided by

MicroCal, using equations and curve-fitting analysis to

obtain least-square estimates of the binding enthalpy,

stoichiometry, and binding constant. Binding stoichio-

metries were derived on the assumption that proteins and

ligand were fully active with respect to binding.

Fluorescence measurements

Binding of the nucleotide was monitored by changes in the

intrinsic trytophan fluorescence of Spo0B. Experiments

were performed in a 1 mL fluorimeter cuvette at 25 �Cusing a Fluoromax 4 spectrofluorimeter [57,58]. The excita-

tion wavelength was 290 nm (slit width 5 nm), and emission

was observed between 300 and 450 nm (slit width 5 nm).

Spo0B was diluted to 375 nm in 1 mL of buffer containing

20 mm Tris ⁄HCl (pH 7.5), 100 mm KCl, and 5 mm MgCl2,

supplemented with increasing concentrations of ATP. All

spectra were corrected for buffer fluorescence and for dilu-

tion (never exceeding 2% of the original volume). Dissocia-

tion constants (Kd) for Mg-ATP binding to Spo0B were

determined by fitting of a hyperbolic plot to the titration

data.

Acknowledgements

Financial support to A. R. Mattoo, M. Saif Zaman,

G. P. Dubey, A. Arora and A. Narayan from the

Council of Scientific and Industrial Research (CSIR) is

acknowledged. The project was supported by CSIR



project NWP-0038. We would like to thank Dr V. C.

Kalia and Dr B. L. Jailkhani for helpful suggestions.

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Supplementary material

The following supplementary material is available

online:

Fig. S1. (A) Comparison of spo0B gene locus of Bacil-

lus anthracis with its orthologs. (B) Amino acid and

nucleotide sequence of Spo0B of Thermoanaero-

bacter tengcongensis.

Fig. S2. Multiple sequence alignment of Spo0B from

different Bacillus species.

Fig. S3. Autophosphorylation of the histidine mutants

H87, H91 and H109 of Spo0B.

Table S1. Distribution of Spo0B and its homologs.

Table S2. Site-specific primers for the mutagenesis of

six histidines and a lysine of Spo0B.

This material is available as part of the online article

from http://www.blackwell-synergy.com

Please note: Blackwell Publishing are not responsible

for the content or functionality of any supplementary

materials supplied by the authors. Any queries (other

than missing material) should be directed to the corre-

sponding author for the article.



Spo0B of Bacillus anthracis – a protein with pleiotropic functions

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