JOURNAL OF BACTERIOLOGY, July 2006, p. 5299–5303
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 188, No. 14
Phosphate Starvation Induces the Sporulation Killing
Factor of Bacillus subtilis
Nicholas E. E. Allenby,1Carys A. Watts,2Georg Homuth,2† Zolta ´n Pra ´gai,2‡ Anil Wipat,3
Alan C. Ward,1and Colin R. Harwood2*
School of Biology and Physcology,1Institute for Cell and Molecular Biosciences,2and School of Computing Science,3
University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, United Kingdom
Received 17 January 2006/Accepted 2 May 2006
Bacillus subtilis produces and exports a peptide sporulation killing factor (SkfA) that induces lysis of sibling
cells. skfA is part of the skf operon (skfA-H), which is responsible for immunity to SkfA, as well as for
production and export of SkfA. Here we report that transcription of skfA is markedly induced when cells of B.
subtilis are subjected to phosphate starvation. The role of PhoP in regulation of the skf operon was confirmed
by in vitro gel shift assays, which showed that this operon is a new member of the PhoP regulon. A putative
stem-loop structure in the skfA-skfB intergenic region is proposed to act as a stabilizer of an skfA-specific
Phosphate starvation leads to marked changes in the expres-
sion of genes in the PhoP and SigB (?B) regulons of Bacillus
subtilis (2, 4, 8, 12, 16, 27). Genes in the PhoP regulon provide
a specific response to phosphate starvation stress, while genes
in the SigB regulon provide a general response to the resulting
energy stress. Additionally, the PhoP and SigB regulons inter-
act to modulate the level to which each regulon is activated (4,
27). The activity of the PhoP regulon is also influenced by
Spo0A; activated Spo0A (Spo0A?P) is responsible for the
induction of sporulation and the repression of genes induced
by the transition-phase regulator AbrB. The PhoP regulon is
up-regulated in a spo0A null mutant that is unable to initiate
sporulation (18, 25). Maximal induction of the PhoP regulon
also requires an active ResD-ResE respiration signal transduc-
tion system (16). An spo0A abrB resD null mutant is not able to
mount a specific response to phosphate starvation, showing
that the induction of the PhoP regulon is dependent not only
on the phosphate-specific PhoPR signal transduction system
but also on this network of regulatory elements (30). If, despite
these regulatory responses, phosphate starvation persists,
Spo0A initiates sporulation and terminates the phosphate re-
sponse by repressing the transcription of phoPR via AbrB and
ResD-ResE (16, 17).
During the phosphate starvation-specific response, genes of
the PhoP regulon are regulated by the PhoP-PhoR two-com-
ponent signal transduction system (2, 25, 29). The PhoP re-
sponse regulator is activated by its cognate sensor kinase,
PhoR. Phosphorylated PhoP (PhoP?P) induces the expression
of the phoPR operon about threefold from a low constitutive
level of expression (17, 26, 27) and is required for the induction
or repression of other members of the PhoP regulon (16).
Fawcett et al. (9) and Molle and colleagues (21) have shown
that Spo0A regulates the skf operon, which encodes the sporu-
lation killing factor (SkfA). SkfA induces the lysis of sibling B.
subtilis cells that have not entered the sporulation pathway
(i.e., Spo0A inactive), providing a source of nutrients to sup-
port this key differentiation process.
Reporter gene analysis of the response of skfA to phosphate
starvation. Previous DNA array analysis of the response of B.
subtilis to phosphate starvation indicated that skfA was induced
in response to phosphate starvation in a phoR-dependent man-
ner (2). This induction was transient, and the level typically
returned to a noninduced level within 3 h. The induction of
skfA in response to phosphate starvation was confirmed with
strain 168-SKFA (Table 1), in which skfA is transcriptionally
fused to a lacZ reporter (skfA::lacZ). Cultures of 168-SKFA
grown in LPM (0.42 mM Pi) were sampled to monitor the
alkaline phosphatase (APase) and ?-galactosidase activities
(24, 27). Three independent experiments gave comparable re-
sults, and a representative data set is shown in Fig. 1. APase, an
intrinsic reporter of the PhoP regulon, was induced during the
transition from the exponential phase to stationary phase (T0),
confirming that entry into stationary phase was due to phos-
phate starvation. skfA was induced (?60 to 70 nmol of o-
nitrophenol [ONP]/min/unit of optical density at 600 nm
[OD600]) concomitantly with APase, as determined by the
?-galactosidase activity of the reporter strain (Fig. 1).
To analyze skfA gene expression in a phoR null background,
B. subtilis 168-SKFA was transformed with chromosomal DNA
from 168-PR (a phoR null mutant) (26) to obtain strain 168-
SKFA-PR. Strain 168-SKFA-PR was used to determine whether
the response of skfA to phosphate starvation was dependent on
the PhoPR regulatory system. Compared to the pattern of lacZ
expression in 168-SKFA, the pattern of lacZ expression in 168-
SKFA-PR was markedly different; low levels of ?-galactosidase
activity (?10 nmol ONP/min/OD600unit) were observed during
the transition phase, but the level increased to ?50 nmol ONP/
* Corresponding author. Mailing address: Institute for Cell and Mo-
lecular Biosciences, University of Newcastle upon Tyne, Newcastle
upon Tyne, NE2 4HH, United Kingdom. Phone: 44 (0)191 222-7708.
Fax: 44 (0)191 222-7736. E-mail: email@example.com.
† Present address: Center for Functional Genomics, Ernst Moritz
Arndt University Greifswald, Medical School, Walther-Rathenau-Str.
49A, D-17489 Greifswald, Germany.
‡ Present address: DSM Nutritional Product Ltd., Department of
Biotechnology, VFB, Bldg. 203/24A, CH-4002 Basel, Switzerland.
min/OD600unit at 6 h after entry into stationary phase (T6) (Fig.
Since skfA is a member of the Spo0A regulon (21), in order
to determine whether Spo0A had a role in the induction of
skfA during phosphate starvation, a spo0A null mutation was
introduced into strain 168-SKFA (skfA::lacZ) (5). The spo0A
null lesion eliminated the transcription of skfA but not the
transcription of another member of the PhoP regulon, phoA,
since alkaline phosphatase was induced normally during the
transition and during early stationary phase (Fig. 1).
Transcription of skfA in response to phosphate starvation.
Northern blot analysis (14, 25) was used to monitor the ex-
pression of genes in the skf operon in response to phosphate
starvation, using RNA extracted from the wild type and a phoR
null mutant as described previously (7). The extracted RNA
was probed with antisense RNA specific for sequences in the
skfA, skfB, and skfH genes. Probes were prepared by PCR with
primer pairs (Table 1) by incorporating a promoter sequence
recognized by T7 polymerase into the reverse primer. The
resulting amplicons were used as substrates for in vitro T7
RNA polymerase-directed synthesis of digoxigenin-labeled skf-
specific RNA probes (1).
Irrespective of the strain, little or no skf-specific RNA was
detected in samples taken 2 h before the onset of phosphate
starvation (Fig. 2), confirming the low level of expression of
this operon under phosphate-replete conditions (Piconcentra-
tion, ?0.1 mM). When the skfA-specific probe was used, a very
intense ?0.25-kb transcript was detected during the transition
to phosphate limitation (T0) and 5 h later (T5) in the wild-type
strain (Fig. 2A). The size of this transcript is consistent with the
FIG. 1. Growth and reporter gene activities of B. subtilis skfA-lacZ
reporter gene fusion strains grown in LPM. (A) OD600of mutants
168-SKFA-PR (phoPR null mutant) (F), 168-SKFA (}), and 168-
SKFA-OA (spo0A null mutant) (■) and APase activities of strains
168-SKFA ( ? ), 168-SKFA-OA (?), and 168-SKFA-PR (E). (B) Tran-
scriptional activities of skfA-lacZ: specific ?-galactosidase activities in
mutants 168-SKFA-PR (F), 168-SKFA-OA (■), and 168-SKFA (}).
TABLE 1. Bacterial strains, plasmids, and primers
Relevant characteristic(s) or sequence (5?–3?)a
B. subtilis strains
trpC2 skfA::pYBCOdd Emr(previously YBCOdd)
T. Tanaka, Takai
T. Tanaka, Takai
trpC2 phoR?BalI::TcrskfA::pYBCOdd Emr
trpC2 pheA1 spo0A::Kmr
trpC2 spo0A::KmrskfA::pYBCOdd Emr
trpC2 spo0A::KmrphoR?BalI::TcrskfA::pYBCOdd Emr
trpC2 skfB::pYBCPdd Emr(previously YBCPdd)
pET2816 containing a 722-bp insert of phoP Apr(6.386 kb)
pET2816 containing a 1,049-bp insert of phoR Apr(6.713 kb)
aSequences in boldface type are specific for the T7 promoter used to initiate in vitro transcription.
bPositions of the primers specific for the B. subtilis 168 chromosome as indicated in the SubtiList database (http://genolist.pasteur.fr/SubtiList) (22).
5300 NOTESJ. BACTERIOL.
predicted length of skfA. In the case of the phoR null mutant,
an ?0.25-kb skfA-specific transcript was detected at T5, but
little or no transcript was detected at T0, confirming that the
expression of this gene during phosphate starvation-induced
entry into the transition phase is under the influence of the
PhoPR two-component signal transduction system. When the
gels were run long enough to remove the ?0.25-kb transcript
(the intensity of which obscured a significant portion of the
gel), a larger less intense transcript was observed (Fig. 2B).
The size of this transcript, ?6.5 kb, was consistent with the
hypothesis that it carried the entire skfA-H operon. When
hybridization reactions were carried out with skfB- and skfH-
specific probes, a similar ?6.5-kb transcript was observed (Fig.
2B), but the ?0.25-kb transcript was not observed. Additional
bands at intermediate sizes could have been either processing
products or mRNA entrapped in the rRNA bands (1).
DNA microarray analysis (11) of the mRNA decay rates of
?1,500 B. subtilis mRNA transcripts in early-stationary-phase
cultures indicated that about 80% of them had a half-life of
less than 7 min. However, the skfA transcript was among ?30
mRNA species that were found to have a half-life of ?15 min.
We therefore attempted to determine the half-life of the phos-
phate starvation-induced skfA-specific transcript following
treatment with rifampin, an inhibitor of transcription initia-
tion. B. subtilis was grown in LPM, and rifampin was added to
the culture 2 h after the transition to the phosphate starvation-
induced transition phase (T2). Samples were harvested imme-
diately before the addition of rifampin (zero time) and at 2, 10,
25, and 45 min after the addition of the antibiotic. The samples
were hybridized with the skfA-specific probe. As shown in Fig.
2C, the skfA mRNA transcript was extremely stable, and no
observable decrease in the amount of transcript was detected
45 min after the addition of rifampin. Analysis of the skfA-skfB
intergenic region using the MFOLD program (32) predicted
that the RNA in this region is able to form a stable stem-loop
structure (?G ? ?23.3 kcal/mol) (data not shown).
Binding of PhoP and PhoP?P to the region of the skfA
promoter. The B. subtilis genome sequence was interrogated us-
ing the pattern-matching function of SubtiList (http://genolist
.pasteur.fr/SubtiList/) (22) to identify potential PhoP binding se-
quences. A 1-bp deviation from the PhoP consensus sequence
(TTHACA3-7TTHACA, where H is A, C, or T) was allowed for
each element of the search sequence, and only targets within 200
bp of a start codon were reported. The promoter region of skfA
was found to contain at least one PhoP consensus sequence, and
a possible second sequence was located immediately downstream
(data not shown).
To determine whether the putative PhoP binding sequences
identified were active in vitro, gel shift assays we used to ana-
lyze the binding of PhoP to the promoter region of skfA.
Proteins PhoP-His6and PhoR231-His6were produced from
Escherichia coli BL21(?D3) carrying pET-PhoP or pET-
PhoR231 (25), as described previously (6). Fragments of the
skfA and yhaX promoter regions were amplified with primers
YbcO-FOR and YbcO-REV and with primers YhaX-FOR
and YhaX-REV (Table 1). In the gel shift reactions, 4 ?M
PhoR231 and 0.1 ?g of poly(dI-dC) (Sigma) per ?l were in-
cubated with 0, 24, 47, 71, and 95 ?M PhoP in the presence or
absence of 5 mM ATP for 15 min at room temperature (20).
After addition of the DNA the mixture was incubated for a
further 30 min. The samples were analyzed on a 6% native
polyacrylamide gel by using Tris-glycine buffer (28) and were
stained with Sybr GOLD (Molecular Probes).
Gel shift assays showed that both PhoP and PhoP?P de-
creased the mobility of the DNA fragment encoding the skfA
promoter region (Fig. 3A). As described previously (25), there
was very little difference in the observed retardation of the
probe when PhoP was phosphorylated, indicating either that
PhoP and PhoP?P bind to the skfA promoter region in vitro
with similar efficiencies or that PhoP is spontaneously phos-
phorylated in E. coli (19). As a negative control, a gel shift
FIG. 2. (A) Northern blot analyses of the skf operon. Total RNA
was isolated from wild-type strain B. subtilis 168 and a phoR null
mutant. Bacteria were grown in LPM, and samples were taken 2 h
before (T?2), at the time of (T0), and 5 h after (T5) entry into station-
ary growth phase provoked by phosphate starvation. Five micrograms
of RNA was applied to each lane, and then after capillary blotting the
filters were hybridized to skfA gene-specific riboprobes. (B) Northern
blot analyses of the skf operon. RNA was isolated from wild-type strain
B. subtilis 168. Bacteria were grown in LPM, and samples were taken
2 h before (T?2), at the time of (T0), and 2 h after (T2) entry into
stationary growth phase provoked by phosphate starvation. Ten mi-
crograms of RNA was applied per lane, and then after an alkaline-
transblotting procedure the filters were hybridized to gene-specific
riboprobes for skfA, skfB, and skfH. The 0.25-kb transcript was pur-
posely run off the bottom of the gel to facilitate visualization of the
weaker bands. (C) Transcriptional stability of skfA mRNA. RNA was
isolated from wild-type strain B. subtilis 168, grown in LPM, and
sampled 2 h after entry into stationary growth phase. RNA was iso-
lated from samples harvested before and 2, 10, 25, 45 min after the
addition of rifampin. Five micrograms of RNA was applied to each
lane, and the membrane was hybridized to a skfA-specific riboprobe.
Transcript sizes were determined by comparison with digoxigenin-labeled
RNA size markers (Roche Diagnostics, Mannheim, Germany). An RNA
molecular size ladder (0.24 to 9.5 kb) was purchased from Invitrogen.
VOL. 188, 2006NOTES 5301
assay was performed for the promoter region of yhaX (Fig.
3B), a gene that appears to be induced indirectly by PhoP. As
shown previously (25), the mobility of yhaX was not influenced
Conclusions. B. subtilis has a range of adaptive responses
which are induced under adverse conditions to maintain via-
bility and, in some cases, to restore growth (1, 2, 4, 15). The
role of SkfA is to bring about the lysis of cells that have not
been induced to enter the sporulation pathway. This provides
cells with activated Spo0A (Spo0A?P) with a source of nutri-
ents (including phosphorus from the cell wall, nucleic acids,
membranes, and proteins) (9, 10, 21). The induction of skfA
during the phosphate starvation-induced transition phase (T0
to T3) is dependent on PhoPR. This response is transient and
is presumably terminated by Spo0A (18). A later induction
event (T3to T6) (Fig. 2) is PhoPR independent and likely to be
associated with the onset of sporulation since skfA is a member
of the Spo0A regulon (9, 21).
The binding of PhoP to the skfA promoter region was com-
pared with the binding to the SigE-dependent yhaX promoter
region (25). In contrast to the binding to yhaX, PhoP binds
directly to the promoter region upstream of skfA, indicating
that the skf operon is a member of the PhoP regulon. Northern
blot hybridization (Fig. 2) detected a weak band corresponding
to a full-length transcript of the skf operon (6.5 kb) and a much
stronger band for the ?0.25-kb transcript corresponding to
skfA. Our results extend previous data (11) showing that the
skfA transcript is extremely stable, with an estimated half-life
of more than 45 min. Since the kinetics of induction and the
relative amounts of ?-galactosidase synthesized by strains 168-
SKFA (skfA::lacZ) (Fig. 1) and 168-SKFB (skfB::lacZ) (not
shown) in response to phosphate starvation were similar, it is
likely that the genes are transcribed from the same promoter.
By analogy with the pst operon (1), we propose that the stem-
loop acts primarily as a barrier to 3?-5? exoribonuclease activity
with the skfA transcript and is therefore responsible for its
extraordinary stability (13, 23). This provides a mechanism for
coordinating the transcription of all the genes in the skf
operon, while it allows preferential translation of skfA mRNA.
Functionally, this should allow a large molar excess of the
killing factor peptide compared with the other products of the
Phosphate limitation is a common feature of life in the soil,
the natural environment of B. subtilis. Induction of the sporu-
lation killing factor by cells that have entered the sporulation
pathway and the subsequent lysis of the noninduced portion of
the population provide the former organisms with an impor-
tant source of nutrients. The resulting influx of nutrients in-
creases the survival potential of these cells and delays the onset
of sporulation (9, 10, 21). We showed that the skf operon is
induced in response to phosphate starvation in a PhoPR-de-
pendent manner. This induction is distinct from that previously
reported for Spo0A. An interesting aspect of this induction is
its absolute dependence on Spo0A. This observation reinforces
previous data which showed that the presence of fully func-
tional PhoPR is not sufficient for maximal expression of the
PhoP regulon, since an spo0A abrB resD null mutant does not
show PhoP regulon activity (16). In the case of the skf operon,
the requirement for Spo0A presumably arises from the need to
coordinate its expression with sporulation since the sporulation
killing factor’s role is to provide nutrients for the subpopula-
tion of cells that are committed to this differentiation process.
We thank T. Tanaka (Tokai University, Japan) for the gift of strain
This work was funded by the European Commission (grant QLG2-
CT-1999-01455) and the UK Biotechnology and Biological Sciences
Research Council (grant 13/PRES/12179). N.E.E.A. was a recipient of
a studentship from the UK Biotechnology and Biological Sciences
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temperature for 15 min. skfA (A) and yhaX (B) promoter probes were
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