JOURNAL OF BACTERIOLOGY, Oct. 2008, p. 6668–6675
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 190, No. 20
Identification of Residues Important for Cleavage of the Extracellular
Signaling Peptide CSF of Bacillus subtilis from Its Precursor Protein?†
Sara Lanigan-Gerdes,1Geraldine Briceno,1Alek N. Dooley,2
Kym F. Faull,2,3and Beth A. Lazazzera1,3*
Department of Microbiology, Immunology and Molecular Genetics,1The Pasarow Mass Spectrometry Laboratory, The NPI-Semel Institute for
Neuroscience and Human Behavior and Department of Psychiatry and Biobehavioral Sciences,2and Molecular Biology Institute,3
University of California at Los Angeles, Los Angeles, California 90095
Received 2 July 2008/Accepted 30 July 2008
Extracellular Phr pentapeptides produced by gram-positive, spore-forming bacteria regulate processes
during the transition from exponential- to stationary-phase growth. Phr pentapeptides are produced by
cleavage of their precursor proteins. We determined the residues that direct this cleavage for the Bacillus
subtilis Phr peptide, CSF, which is derived from the C terminus of PhrC. Strains expressing PhrC with
substitutions in residues ?1 to ?5 relative to the cleavage site had a defect in CSF production. The mutant
PhrC proteins retained a functional signal sequence for secretion, as assessed by secretion of PhrC-PhoA
fusions. To determine whether the substitutions directly affected cleavage of PhrC to CSF, we tested cleavage
of synthetic pro-CSF peptides that corresponded to the C terminus of PhrC and had an amino acid substitution
at the ?2, ?3, or ?4 position. The mutant pro-CSF peptides were cleaved less efficiently to CSF than the
wild-type pro-CSF peptide whether they were incubated with whole cells, cell wall material, or the processing
protease subtilisin or Vpr. To further define the range of amino acids that support CSF production, the amino
acid at the ?4 position of PhrC was replaced by the 19 canonical amino acids. Only four substitutions resulted
in a >2-fold defect in CSF production, indicating that this position is relatively immune to mutational
perturbations. These data revealed residues that direct cleavage of CSF and laid the groundwork for testing
whether other Phr peptides are processed in a similar manner.
Gram-positive bacteria secrete small peptides into their en-
vironment that are used to self-monitor population density
and/or the diffusivity of the environment, processes that are
referred to as quorum sensing (3, 11, 19). The Phr peptides are
pentamers that are secreted by gram-positive, endospore-
forming bacteria to mediate quorum sensing or to control the
timing of gene expression (26, 28). While much is known about
the mechanisms involved in sensing and responding to the Phr
peptides, there are still questions regarding the production of
The Phr signaling peptides were first identified in Bacillus
subtilis, where their functions include control of the develop-
ment of genetic competence (the ability to take up exogenous
DNA), sporulation (the formation of environmentally resistant
spores), excision and transfer of a mobile DNA element, and
production of extracellular degradative enzymes (2, 23, 26, 28).
In Bacillus anthracis, Phr signaling peptides regulate sporula-
tion, and in Bacillus cereus and Bacillus thuringiensis, a Phr-type
signaling peptide regulates expression of virulence genes (5,
29). The functions of putative Phr signaling peptides encoded
by the genomes of other bacteria have not been characterized
The B. subtilis Phr peptide, CSF, is a prototypical Phr pep-
tide. This pentameric peptide (sequence, ERGMT) is derived
from the C terminus of the PhrC precursor protein (31). PhrC
has an N-terminal signal sequence for export through the Sec-
dependent export pathway (28). When it is extracellular, PhrC
is processed by one of three redundant proteases, subtilisin,
Epr, or Vpr, to release CSF (15). At a critical extracellular
concentration, CSF is transported into the cell by an oligopep-
tide permease (17). Once it is inside the cell, CSF interacts
with cytoplasmic receptor proteins, RapC and RapB, to inhibit
their activity (7, 25). RapC binds to and inhibits the DNA-
binding activity of the ComA transcription factor (7), which
regulates the expression of genes involved in extracellular and
membrane functions, as well as genetic competence develop-
ment (6, 24). By inhibiting RapC, CSF stimulates expression of
ComA-controlled genes. ComA-controlled gene expression is
similarly stimulated by several other Phr peptides, including
PhrF, PhrG, PhrH, and PhrK (1, 4, 10, 30). However, CSF also
inhibits ComA-controlled gene expression at higher concen-
trations by an incompletely understood mechanism (4, 16, 17).
RapB, the other identified cytoplasmic receptor for CSF, de-
phosphorylates Spo0F, a response regulator protein required for
sporulation (34). RapC, RapB, and the other identified cytoplas-
mic receptor proteins of Phr peptides are all members of the
tetratricopeptide repeat domain family of proteins (7, 26).
Some Phr signaling peptides are derived from the C termini
of their precursor proteins, whereas others are derived from
internal portions (12, 31). The identity of the determinants of
the cleavage site for release of the Phr pentapeptides is an
important unanswered question. To address this question for
B. subtilis Phr peptides, we previously aligned the B. subtilis Phr
precursor proteins based on the known or predicted mature
* Corresponding author. Mailing address: 1602 Molecular Sciences
Bldg., 405 Hilgard Ave., Los Angeles, CA 90095. Phone: (310) 794-
4804. Fax: (310) 206-5231. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://jb
?Published ahead of print on 8 August 2008.
pentapeptide sequences (28). A loose consensus sequence was
identified; this sequence was not a strict amino acid sequence
but consisted of a string of amino acids with particular chem-
ical characteristics. It was located at the five residues (residues
?5 to ?1) preceding the cleavage site; however, for 3 of the 13
Phr proteins insertion of a one-residue gap was necessary for
alignment (Table 1). We hypothesized that these five residues
could be important for directing the cleavage event. Consistent
with this hypothesis, amino acid substitutions at the ?1 and ?3
positions relative to the cleavage site for PhrA, PhrE, and CSF
decreased the expression of genes controlled by these Phr
peptides (33). Here, we demonstrate that changes in any of the
five residues preceding the cleavage site in PhrC reduced CSF
production and directly affected cleavage of synthetic pro-CSF
MATERIALS AND METHODS
Growth conditions. B. subtilis cells were grown with shaking at 37°C in S7
minimal medium (35), except that the concentration of MOPS (morpholinepro-
panesulfonic acid) was 50 mM rather than 100 mM. This medium contained 1%
glucose, 0.1% glutamate, and required amino acids (tryptophan, phenylalanine,
and, when necessary, threonine) at a concentration of 50 ?g/ml. When appro-
priate, 1 mM isopropyl-?-D-thiogalactopyranoside (IPTG) was added, and anti-
biotics were added at the following concentrations: ampicillin, 100 ?g/ml; chlor-
amphenicol, 5 ?g/ml; erythromycin,0.5 ?g/ml; neomycin, 5 ?g/ml; spectinomycin,
100 ?g/ml; and tetracycline, 12.5 ?g/ml.
Strain and plasmid construction. B. subtilis strains used in this study are listed
in Table 2. The B. subtilis strains were constructed by transformation with
chromosomal DNA or plasmids using standard protocols (9).
Plasmid pBL495, containing the thrC::(Pspachy-phrC cat) allele, was constructed
as follows. First, plasmid pBL354, containing the thrC::cat allele, was constructed
by swapping the erm locus of plasmid pDG1664 (8) with the cat locus of pGEM-
cat (36). All of pDG1664 except the erm locus was PCR amplified using Taq
polymerase (Qiagen) and primers BL322 (5?-CAATTTCGTAATCGGAACGG
TATCGG-3?) and BL323 (5?-CGTTACTAATCGCGAAGGGAATGTAG-3?),
and the cat locus was amplified from pGEM-cat using Vent polymerase (New
England Biolabs) and primers CM1 (5?-AAGCATGCGTTACCCTTATTATC
AAGA-3?) and CM2 (5?-AAGCATGCGGAGCTGTAATATAAAAAC-3?).
The ends of the pDG1664 PCR product were blunted using an end repair kit
(Epicentre, Madison, WI), and then the two PCR products were ligated to
generate plasmid pBL354, in which cat is transcribed in the same direction as
thrC. pBL354 was then digested with EcoRI and BamHI and ligated to an
EcoRI-BamHI fragment containing lacI and Pspachy-phrC from pBL24 (15) to
thrC::(Pspachy-phrC erm) alleles with mutations affecting the five residues pre-
ceding the cleavage site for CSF and thrC::(Pspachy-phrC cat) alleles with muta-
tions affecting residue 32 (position ?4) of phrC were constructed by site-directed
mutagenesis of plasmids pBL24 (15) and pBL495, respectively, using a
QuikChange site-directed mutagenesis kit (Stratagene). The phrC genes were
then sequenced to confirm the presence of the desired mutations. The primers
used for mutagenesis and the designations of the resulting plasmids are shown in
Table S1 in the supplemental material. B. subtilis cells were transformed with
these plasmids, and either erythromycin- or chloramphenicol-resistant transfor-
mants were selected as appropriate. Transformants were checked for replace-
ment of the thrC locus by Pspachy-phrC based on the lack of spectinomycin
resistance associated with the plasmids and auxotrophy for threonine.
thrC::[Pspachy-?(phrC-?phoA) erm] alleles were constructed as follows. A por-
tion of phoA encoding an alkaline phosphatase lacking the N-terminal signal
sequence was PCR amplified from chromosomal DNA of Escherichia coli strain
MC4100 with primers BL474 (5?-GAAACCCGGGTACCGTTACTGTTTACC
C-3?) and BL466 (5?-GGTTAGATCTGCTAACAGCAAAAAAACCACCCG
G-3?) containing SmaI and BglII sites (underlined), respectively. The amplified
phoA gene was cloned into the corresponding sites of pBL112 (15). The various
alleles of the phrC gene were released from pBL24 (15) and cloned into the
HindIII and SmaI sites of the pBL112 plasmid containing phoA. Site-directed
mutagenesis (Stratagene QuickChange mutagenesis kit) was then performed
with primers BL580 (5?-GAGGAATGACGTTTACCCCTGTGACAAAAGCC
CGGACACCAG-3?) and BL581 (5?-CTGGTGTCCGGGCTTTTGTCACAGG
GGTAAACGTCATTCCTC-3?) to translationally fuse phrC and phoA. The con-
structed plasmids containing the phrC-?phoA fusions were designated pBL483 to
A negative control strain was constructed, in which the phrC ribosome-binding
site was fused to a truncated phoA gene, ?(phrCRBS-?phoA), which expressed a
signal- sequence-less alkaline phosphatase. To obtain this construct, phoA was
amplified using reverse primer BL466, which contained a BglII site, and forward
primer BL475 (5?-GCGCAAGCTTAAAGGAGTGAAGGTTTGTATGTACTGT
TTACCC-3?), which contained the 18 bp preceding phrC, including the ribo-
some-binding site (bold type), and a HindIII site (underlined). The PCR product
was cloned into the corresponding sites of pBL112 (15) to generate plasmid
phrC-?phoA fusion constructs of plasmids pBL482 to pBL489 were sequenced
to confirm proper construction. B. subtilis cells were transformed with these
plasmids, and erythromycin-resistant transformants were selected. Transfor-
mants were checked for replacement of the thrC locus with various Pspachy-
?(phrC-?phoA) erm alleles based on the lack of spectinomycin resistance asso-
ciated with the plasmids and auxotrophy for threonine.
Isolation of PhrC-F32 substitution mutants. To isolate phrC mutations that
decreased production of CSF by altering the codon for residue 32 of PhrC (i.e.,
the ?4 position relative to the cleavage site for CSF), we used site-directed
mutagenesis to randomize this codon. Site-directed mutagenesis was performed
on plasmid pBL495 using primers BL597 (5?-CTAATGCGGAAGCACTCGAC
NNNATGTGACAGAAAGAGGAATGACG-3?) and BL598 (5?-CGTCATTC
CTCTTTCTGTCACATGNNNGTCGAGTGCTTCCGCATTAG-3?) (where N
is any base). The resulting site-directed mutagenesis products were transformed
into E. coli XL10-Gold Ultracompetent cells (Stratagene) with selection for
ampicillin-resistant transformants. All transformant colonies were pooled in LB
medium with ampicillin and grown at 37°C for 1 h, and then plasmid DNA was
isolated. Isolated plasmid DNA was passaged though E. coli strain MC1061 (F?
lacIqlacZM15 Tn10) before it was transformed into B. subtilis strain BAL1147
(rapA-lacZ ?phrC), and transformants were selected on agar plates containing
Difco sporulation medium, chloramphenicol, IPTG, and 200 ?g/ml X-Gal (5-
bromo-4-chloro-3-indolyl-?-D-galactopyranoside). After 18 h of incubation,
white colonies were screened for further characterization. Under these growth
conditions, a ?phrC mutant strain was white, and a strain containing a wild-type
copy of phrC was blue (data not shown). Putative phrC mutant strains were
screened for a defect in endogenous CSF production, and the strains that showed
a defect were backcrossed into BAL1147 by selecting for the chloramphenicol
resistance associated with the thrC::Pspachy-phrC cat allele and rechecked for a
TABLE 1. Cleavage sites for B. subtilis Phr peptides
Phr proteinPro sequencea
release of the mature Phr pentapeptides. The consensus sequence for the five
residues preceding the cleavage site is (a/p)h(c/p)VA, where “a” is an acidic or polar
residue, “h” is a hydrophobic residue, and “c” is a charged or polar residue. Bold
type indicates residues that are the same as residues in the consensus sequence.
Underlining indicates residues that are different than residues in the consensus
bThere are amino acids C terminal to the sequence of the mature Phr peptide
in the precursor Phr protein.
cThe PhrH peptide fits the consensus least well, and the prediction for the
mature pentapeptide sequence should be interpreted with caution.
dPredicted sequence of a Phr peptide for which there is not direct experimen-
tal evidence. The predicted Phr peptide was chosen because its sequence was
most similar to the sequence of a known Phr peptide of B. subtilis.
ePhr-pPL10-1 is encoded on a Bacillus pumilus plasmid.
VOL. 190, 2008RESIDUES AFFECTING CSF PROCESSING6669
defect in endogenous CSF production. The phrC gene of 25 mutant strains with
a defect in CSF production was sequenced. BAL3029 with an F32S substitution
(TTT-to-AGT codon change), BAL3030 with an F32G substitution (TTT-to-
GGC codon change), and BAL3031 with an F32R substitution (TTT-to-CGA
codon change) were obtained using this procedure. The remaining sequenced
phrC mutants contained stop codons or nonoptimal usage codons, as determined
in the study of Kanaya et al. (13).
We generated mutants in which residue 32 of PhrC was replaced by the
remaining 15 amino acids that had not been tested yet at this position. Site-
directed mutagenesis was performed with plasmid pBL495 using primers listed in
Table S1 in the supplemental material, and the results were confirmed by se-
quencing. The plasmids generated using this mutagenesis procedure are listed in
Table S1 in the supplemental material and were transformed into BAL1147.
Assay for endogenous CSF production by cells. Cultures were grown to an
optical density at 600 nm (OD600) of 0.2, and then 1 mM IPTG was added to
induce phrC expression. At an OD600of ?0.7, a 7-ml sample was harvested. The
cells were removed by centrifugation, and the culture supernatant was filtered
(0.2 ?m) and stored at ?20°C. CSF in the supernatant was quantified using the
biological assay described previously (15, 16).
In vitro pro-CSF cleavage assay. The following peptides were synthesized by
Bio-Synthesis Incorporated (Lewisville, TX): pro-CSF-WT (NAEALDFHVTE
RGMT), pro-CSF-F32K (NAEALDKHVTERGMT), pro-CSF-H33A (NAEAL
DFAVTERGMT), and pro-CSF-V34E (NAEALDFHETERGMT) (underlining
indicates a substitution compared to the pro-CSF-WT peptide sequence). The
identities and purities of the preparations were checked in-house by mass spec-
trometry. These synthetic peptides were tested for cleavage by either whole cells,
a cell wall fraction, or subtilisin and Vpr as described previously (15). The levels
of CSF produced after incubation of these peptides were assessed by using either
a biological assay or a mass spectrometric assay, both of which have been
described previously (15, 16).
Alkaline phosphatase assays. Alkaline phosphatase (PhoA) activity assays
were carried out essentially as described by Nicholson and Setlow (22). Strains
were grown in minimal medium. IPTG (1 mM) was added when the OD600of the
cultures reached ?0.2. Incubation was continued until the OD600was ?1. Cul-
ture supernatants were harvested by centrifugation and filtered (0.2 ?m). One-
milliliter aliquots were then each mixed with a 1-ml aliquot of freshly prepared
substrate (1 g/liter p-nitrophenylphosphate in 1 M Tris [pH 8.1]). The reaction
mixtures were incubated at room temperature until they were pale yellow. The
TABLE 2. Strains used in this study
Reference or constructionb
amyE::(srfA-lacZ?374 neo) ?phrC::erm
?opp::erm (formerly ?spo0K::erm)
amyE::??(rapA-lacZ)42 erm? (formerly gisA-lacZ)
?opp::erm ?comQ::spc ?phrC::tet
amyE::??(rapA-lacZ)42 erm? ?phrC::tet
?phrC::tet thrC::(Pspachy-phrC erm)
Same as BAL1191 but phrCT35A
Same as BAL1191 but phrCT35K
Same as BAL1191 but phrCV34E
Same as BAL1191 but phrCH33A
Same as BAL1191 but phrCF32K
Same as BAL1191 but phrCD31A
?phrC::tet thrC::?Pspachy-?(phrC-?phoA) erm?
Same as BAL2200 but phrCRBS
Same as BAL2200 but phrCT35A
Same as BAL2200 but phrCT35K
Same as BAL2200 but phrCV34E
Same as BAL2200 but phrCH33A
Same as BAL2200 but phrCF32K
Same as BAL2200 but phrCD31A
amyE::??(rapA-lacZ)42 ermC? ?phrC::tet thrC::(Pspachy-phrC cat)
Same as BAL2223 but phrCF32P
Same as BAL2223 but phrCF32N
Same as BAL2223 but phrCF32E
Same as BAL2223 but phrCF32D
Same as BAL2223 but phrCF32I
Same as BAL2223 but phrCF32Q
Same as BAL2223 but phrCF32L
Same as BAL2223 but phrCF32Y
Same as BAL2223 but phrCF32C
Same as BAL2223 but phrCF32W
Same as BAL2223 but phrCF32H
Same as BAL2223 but phrCF32V
Same as BAL2223 but phrCF32M
Same as BAL2223 but phrCF32S
Same as BAL2223 but phrCF32G
Same as BAL2223 but phrCF32R
Same as BAL2223 but phrCF32A
Same as BAL2223 but phrCF32T
BAL201 ? BAL3763BAL941
Backcrossed mutant from screen
Backcrossed mutant from screen
Backcrossed mutant from screen
aAll strains are derivatives of BAL218 and contain trpC2 pheA1.
bFor strain construction an arrow indicates the direction of transfer of DNA into the recipient strain. The DNA is either genomic DNA from the indicated strain
or the indicated plasmid.
6670LANIGAN-GERDES ET AL. J. BACTERIOL.
reactions were then stopped by addition of 670 ?l of 2 M NaOH, and the
absorbance at 420 nm was recorded. Fresh minimal medium was used as a blank.
Alkaline phosphatase specific activities were calculated as follows: A420/(incuba-
tion time in minutes ? OD600of culture at supernatant harvest time ? volume
of supernatant in milliliters). The level of alkaline phosphatase activity in each
experiment was normalized to the level produced by the strain containing the
wild-type PhrC-PhoA fusion.
RESULTS AND DISCUSSION
The five residues preceding the cleavage site for CSF are
important for CSF production. We sought to determine
whether the conservation in Phr proteins of five residues pre-
ceding the cleavage site for release of the mature Phr pen-
tapeptides was due to a requirement for these residues for
directing the cleavage event. To this end, we constructed mu-
tants with substitutions at positions ?5 through ?1 relative to
the cleavage site in PhrC that releases CSF. Substitutions that
moved a residue away from the identified consensus sequence
were introduced (Fig. 1A). Site-directed mutagenesis of phrC
was performed, and the mutant phrC genes were introduced
into B. subtilis under the control of the IPTG-inducible Pspachy
promoter in strains lacking endogenous phrC. The strains were
grown to mid-exponential phase in minimal media, and then
PhrC expression was induced. After this the cultures were
grown for ?2 cell doublings, and then culture supernatants
were harvested. The levels of CSF that had accumulated in the
culture supernatants were determined. Briefly, the CSF in the
culture supernatants was partially purified using reverse-phase
chromatography to separate CSF from other signaling peptides
that affect ComA-controlled gene expression, and then it was
quantified based on the ability of CSF to induce expression of
a ComA-responsive reporter fusion, srfA-lacZ. Importantly, a
strain lacking CSF had levels of activity either below or at the
limit of detection (Fig. 1B) (15–17, 31, 32), indicating that any
activity that is observed with other strains is CSF dependent.
The presence of charged or polar residues at the ?5 and ?3
positions was predicted based on the consensus sequence, and
we substituted Ala for the Asp and His residues that occur at
these positions in PhrC to remove the charge and polarity.
These substitutions resulted in 40% (P ? 0.006, n ? 4) and
65% (P ? 0.03, n ? 3) decreases in CSF production for the
Asp-to-Ala and His-to-Ala substitutions, respectively (Fig.
1B). These data support the notion that a charged or polar
residue is important at the ?5 and ?3 positions relative to the
cleavage site for CSF, although the ?3 position appears to be
more important. A Phe residue occurs at the ?4 position of
PhrC, and in the strain expressing PhrC with a Lys residue at
this position there was a 95% (P ? 0.0007, n ? 3) reduction in
CSF production (Fig. 1B). These data are consistent with the
prediction based on the consensus sequence that a hydropho-
bic residue is required at this position. A Val residue is pre-
dicted to be important at the ?2 position, and as we reasoned
that other hydrophobic residues might be functional at this
position, we changed the Val residue of PhrC to Glu. Inter-
estingly, in the strain expressing the Glu variant there was only
a 50% (P ? 0.02, n ? 3) reduction in CSF production (Fig.
1B). Thus, while Val appears to be more optimal for CSF
production than Glu, CSF production was surprisingly tolerant
of a change from Val to a charged residue. The findings for the
?1 position of the consensus sequence indicate that an Ala
residue is critical. Intriguingly, a Thr residue is at this position
in PhrC. We replaced this Thr residue by Ala and found that
the strains produced indistinguishable levels of CSF compared
to the strains with wild-type PhrC (P ? 0.8, n ? 3) (Fig. 1B).
The side chains of Ala and Thr are both small and at least
partially hydrophobic. To test whether these side chains are
important for CSF production, we replaced the Thr residue of
PhrC with Lys, an amino acid with a large, charged side chain.
CSF production was tolerant of such a radical change, and in
strains having the Thr-to-Lys substitution there was only a 35%
(P ? 0.04, n ? 3) decrease in CSF production (Fig. 1B).
Although the effects of some of the amino acid substitutions
were relatively small, together, the data obtained indicate that
the five residues that precede the cleavage site in PhrC for CSF
are required for normal CSF production.
The five residues preceding the CSF cleavage site are not
required for a functional signal sequence. A possible explana-
tion for the decrease in CSF production caused by amino acid
substitutions at positions ?1 through ?5 relative to the CSF
cleavage site is that the substitutions affect an extended signal
sequence necessary for secretion. To test this possibility, we
created fusions of the mutant PhrC proteins to the E. coli
alkaline phosphatase protein (PhoA), which lacked its own
signal sequence. The PhrC-PhoA fusions were expressed in B.
subtilis from the thrC locus under the control of the IPTG-
inducible Pspachypromoter. PhrC-PhoA secretion was moni-
tored by assaying the alkaline phosphatase activity in culture
FIG. 1. Substitution of the five residues preceding the CSF cleav-
age site affects CSF production. (A) Amino acid sequence of the
C-terminal 15 residues of PhrC. The five residues preceding the cleav-
age site are indicated by bold italics, and the mature signaling peptide
(CSF) is underlined. The amino acid substitutions in PhrC are indi-
cated below the PhrC sequence. The putative consensus sequence is
indicated above the PhrC sequence; “a” indicates an acidic residue,
“p” indicates a polar residue, “h” indicates a hydrophobic residue, and
“c” indicates a charged residue. (B) Levels of CSF that accumulated in
culture medium for strains BAL1191 (WT), BAL1192 (?phrC),
BAL1187 (D31A), BAL1186 (F32K), BAL1185 (H33A), BAL1184
(V34E), BAL1183 (T35K), and BAL1182 (T35A). For each of three
independent experiments, the CSF levels were normalized to the level
produced by strain BAL1191. The bars indicate the means of the
normalized values, and the error bars indicate the standard errors of
VOL. 190, 2008RESIDUES AFFECTING CSF PROCESSING 6671
supernatants. As expected, a strain expressing PhoA lacking a
signal sequence had no measurable alkaline phosphatase ac-
tivity (Fig. 2). All of the strains expressing mutant PhrC-PhoA
fusion proteins had alkaline phosphatase activities comparable
to that of a strain expressing the wild-type PhrC-PhoA fusion
protein (P ? 0.25, n ? 3) (Fig. 2). These data indicate that all
of the mutant PhrC proteins contained a functional signal
sequence and suggest that a secretion defect was not the cause
of the reduced CSF production by strains expressing the mu-
tant PhrC proteins.
Synthetic pro-CSF peptides with amino acid substitutions
are cleaved less efficiently to CSF. The data described above
indicated that strains expressing PhrC mutant proteins with
amino acid substitutions at positions ?5 through ?1 relative to
the CSF cleavage site produce less CSF and that the reduction
in CSF production was not due to a reduction in secretion of
PhrC. We reasoned that this reduction could have been due to
reduced PhrC protein expression or reduced protease recog-
nition and/or cleavage of PhrC to CSF. We measured the levels
of phrC mRNA for the three mutant strains with the lowest
levels of CSF production (i.e., the mutants with substitutions at
the ?2 to ?4 positions) and observed no significant differences
in the levels of phrC mRNA (see Fig. S1 in the supplemental
material). There is no method to directly test the levels of PhrC
inside cells at this time, and attempts to indirectly measure
PhrC levels using a C-terminal epitope tag have been unsuc-
cessful, possibly due to proteolytic removal of the tag extracel-
lularly (data not shown). Given that the mutant PhrC-PhoA
fusion proteins were expressed at levels similar to the levels of
the wild-type fusion protein, it seemed unlikely that the defect
in CSF production was due to a defect in PhrC expression.
To test directly the possibility that the mutant PhrC proteins
were cleaved less efficiently to CSF, we synthesized peptides
that corresponded to the portion of PhrC predicted to be C
terminal to the signal sequence for secretion. The sequence of
one peptide, pro-CSF-WT, was identical to the sequence of the
C-terminal 15 residues of PhrC, and this peptide has been used
previously in studies of CSF proteolytic processing (15). We
also synthesized peptides that individually had the three amino
acid substitutions that resulted in the greatest defects in CSF
production, pro-CSF-F32K, pro-CSF-H33A, and pro-CSF-
V34E. These peptides differed at the ?2, ?3, or ?4 position
relative to the CSF cleavage site (Fig. 3A).
To test for cleavage of the synthetic pro-CSF peptides to
CSF, the peptides were incubated with washed whole cells of
B. subtilis strain BAL950, which lacks phrC and thus cannot
produce any CSF. BAL950 also lacks the oligopeptide per-
mease responsible for uptake of CSF from media and comQ,
which encodes a protein needed to produce a signaling peptide
with activity similar to that of CSF. All three of these mutations
were included to increase the sensitivity of the assay for de-
tection of CSF. Approximately 108cells were incubated with
pro-CSF for 40 min, a time sufficient for nearly complete cleav-
age of 100 pmol of pro-CSF-WT to CSF (15). After incubation,
the cells were removed, and the culture medium was fraction-
ated on a reverse-phase C18column to separate pro-CSF from
CSF. The amount of CSF that eluted from the column was
then determined by determining the level of ?-galactosidase
FIG. 2. Mutant PhrC proteins are secreted at the same level as
wild-type PhrC as measured by alkaline phosphatase activity for strains
carrying PhrC-PhoA fusions. PhoA activity was measured using culture
supernatants of strains BAL2199 (?ss-PhoA), BAL2200 (WT), BAL2201
(T35A), BAL2202 (T35K), BAL2203 (V34E), BAL2204 (H33A),
BAL2205 (F32K), and BAL2206 (D31A). The normalized, mean levels of
alkaline phosphatase activity from three independent experiments (indi-
cated by bars) are plotted versus the strains assayed. The error bars
indicate the standard errors of the means.
FIG. 3. Synthetic pro-CSF peptides with amino acid substitutions
at the ?4, ?3, and ?2 positions are cleaved less efficiently to mature
CSF. (A) Sequence of synthetic pro-CSF peptides. (B) Synthetic pro-
CSF peptides were incubated with cells of strain BAL950 (?opp
?comQ ?phrC), and the amount of CSF produced was determined
using the biological assay. The normalized mean amounts of CSF
produced in three independent experiments are indicated by bars, and
the error bars indicate the standard errors of the means. (C) The
pro-CSF peptides were incubated with cell wall material, and
the ERGMT was quantified by LC-MS/MS–MRM. The intensity of the
signal for the parent-to-fragment ion (m/z 386.5) transition is plotted
versus the elution time for C18columns. WT, pro-CSF-WT.
6672 LANIGAN-GERDES ET AL.J. BACTERIOL.
specific activity after treatment of cells containing the ComA-
controlled reporter fusion srfA-lacZ with dilutions of the elu-
When the cells were incubated with pro-CSF-WT, CSF pro-
duction was observed, but CSF production was not observed
when the cells and pro-CSF-WT were incubated separately
(Fig. 3B and data not shown). Compared to pro-CSF-WT, each
of the mutant pro-CSF peptides produced less CSF when it was
incubated with cells (Fig. 3B). Incubation with the pro-CSF-
F32K peptide, with a substitution at the ?4 position, yielded
only 10% of the CSF produced with the pro-CSF-WT peptide
(P ? 0.01, n ? 3), similar to the results obtained with the same
amino acid substitution in the in vivo context (compare Fig. 3B
and 1B). Incubation with the pro-CSF-V34E and pro-CSF-
H33A peptides yielded 25% (P ? 0.004, n ? 3) and 60% (P ?
0.02, n ? 3) of the CSF produced with the pro-CSF-WT pep-
tide, respectively. The magnitude of the defect caused by these
substitutions was different than the magnitude observed when
the same substitutions were encoded by phrC (compare Fig. 3B
and 1B). This may have been because there was a slightly
different profile of proteases able to cleave pro-CSF to CSF
after cells were washed. Nevertheless, the defect in CSF pro-
duction caused by the amino acid substitutions at positions ?2
to ?4 when they were part of an exogenously added peptide
supports the hypothesis that these amino acid substitutions
decreased the efficiency of cleavage of PhrC to CSF.
To confirm that the mutant peptides were cleaved less effi-
ciently to the CSF peptide sequence ERGMT, we measured
the amounts of CSF produced after incubation of the pro-CSF
peptides using a liquid chromatography-tandem mass spec-
trometry (LC-MS/MS) assay for CSF. This assay recorded the
intensity of the transition of the doubly charged parent ion
(m/z 297.2) to the singly charged fragment ion (m/z 386.5)
under collisionally activated dissociation, multiple-reaction-
monitoring (MRM) conditions, as previously described (15).
Because whole bacterial cells would have interfered with the
LC-MS/MS–MRM procedure, we used a cell wall-enriched
fraction of B. subtilis cells, which we have previously shown to
be the cellular fraction that contains the majority of pro-CSF
processing activity (15). Under the prescribed chromatography
conditions, the retention time of synthetic CSF was 14.5 min.
Neither the pro-CSF peptides nor the cell wall fraction of B.
subtilis separately resulted in a significant LC-MS/MS–MRM
response for CSF (data not shown). In contrast, when the cell
wall fraction was incubated with pro-CSF-WT, a significant
MRM response was recorded at the appropriate retention time
(Fig. 3C), indicating that pro-CSF-WT was cleaved into the
CSF pentapeptide. When the cell wall fraction was incubated
with the mutant pro-CSF peptides, the level of the MRM
response was not more than 10% of the level observed with
pro-CSF-WT (Fig. 3C). The greater defect in cleavage of pro-
CSF to CSF for the mutant peptides determined by this assay
than by the biological assays could have been due to the change
in the profile or levels of pro-CSF processing proteases that
occurred during preparation of the cell wall-enriched fraction
of cells. The data obtained demonstrate that the amino acid
substitutions at the ?2, ?3, and ?4 positions significantly
decreased proteolytic cleavage of ERGMT from a precursor
CSF processing proteases, subtilisin and Vpr, cleave mutant
pro-CSF peptides less efficiently. We previously showed that
cells lacking the secreted serine proteases subtilisin, Vpr, and
Epr had a defect in production of CSF and that purified sub-
tilisin and Vpr were able to cleave synthetic pro-CSF to CSF
(15). To further support the hypothesis that subtilisin and Vpr
have a direct role in processing pro-CSF to CSF in vivo, we
examined whether the amino acid substitutions in PhrC that
decreased production of CSF in vivo similarly affected the
cleavage of pro-CSF by subtilisin or Vpr. Purified subtilisin and
Vpr were incubated separately with pro-CSF substrates having
substitutions at positions ?2 to ?4 relative to the cleavage site,
and the levels of CSF produced were determined using the
biological assay for CSF (Fig. 4).
The Phe-to-Lys substitution at the ?4 position resulted in
severely reduced cleavage of pro-CSF (98% reduction for sub-
tilisin [P ? ?0.0001, n ? 3] and 90% reduction for Vpr [P ?
0.001, n ? 3]). The His-to-Ala substitution at the ?3 position
had a more modest effect on cleavage (47% [P ? 0.03, n ? 3]
and 79% [P ? 0.003, n ? 3] reductions in pro-CSF cleavage by
subtilisin and Vpr, respectively). The Val-to-Glu substitution
at the ?3 position had the most disparate effects on pro-CSF
cleavage (97% [P ? ?0.0001, n ? 3] reduction in cleavage to
CSF by subtilisin and 42% [P ? 0.02, n ? 4] reduction in
cleavage by Vpr). Collectively, these data indicate that substi-
tutions at positions ?2 to ?4 decreased the efficiency of cleav-
age of pro-CSF to CSF by subtilisin and Vpr, and they support
the hypothesis that subtilisin and Vpr have a direct role in
processing CSF in vivo.
Defining the amino acids that are tolerated at the ?4 posi-
tion of pro-CSF and allow cleavage. To begin to determine the
rules that govern what amino acid sequences can be recognized
for cleavage that releases mature Phr pentapeptides, we
changed the amino acid at the ?4 position of pro-CSF to the
other 19 canonical amino acids in order to determine which
amino acids support cleavage that releases CSF. The ?4 po-
sition was chosen for this analysis as substitutions at this posi-
tion resulted in the greatest defects in CSF production. CSF
production by strains individually expressing 1 of the 19 mutant
PhrC-F32 proteins was assessed using the biological assay
FIG. 4. Cleavage of mutant pro-CSF substrates by subtilisin and
Vpr. Purified subtilisin or Vpr was incubated with the pro-CSF sub-
strates indicated. The level of CSF produced was normalized to the
level of CSF produced after incubation with the wild-type pro-CSF
substrate (WT). The bars indicate the averages of at least three inde-
pendent experiments, and the error bars indicate the standard errors of
the means. Under these conditions, incubation of subtilisin with pro-
CSF and incubation of Vpr with pro-CSF resulted in statistically in-
distinguishable levels of CSF production.
VOL. 190, 2008RESIDUES AFFECTING CSF PROCESSING6673
The identified consensus sequence of the residues that pre-
cede the cleavage site for Phr peptides indicated that a hydro-
phobic residue is important at the ?4 position. Consistent with
this, the hydrophobic residues, such as Val, Ile, and Leu, sup-
ported levels of CSF production similar to the levels exhibited
by the wild-type strain with a Phe residue at the ?4 position.
Furthermore, substitution of a hydrophobic Met residue at this
position resulted in levels of CSF production that were 1.8-
fold-higher than the levels observed with the Phe residue (P ?
0.03, n ? 4). The only exceptions to this were the hydrophobic
Ala and Trp substitutions, which resulted in 20% (P ? 0.03,
n ? 3) and 50% (P ? 0.03, n ? 3) decreases in CSF produc-
tion, respectively. Trp is the largest amino acid, and the defect
in CSF production may have been due to the large side chain
sterically hindering cleavage. At the other extreme, Ala is the
smallest hydrophobic amino acid, and the defect in CSF pro-
duction may have been due to the small size of the side chain,
which was not able to stabilize the interaction with pro-CSF
As further support for the hypothesis that a hydrophobic
residue at the ?4 position is important, some polar residues at
this position decreased production of CSF. As noted above, a
Lys substitution severely decreased CSF production; in addi-
tion, Arg, Cys, and Asp substitutions decreased CSF produc-
tion 72% (P ? 0.05, n ? 3), 68% (P ? 0.008, n ? 3), and 44%
(P ? 0.03, n ? 5), respectively. However, some polar amino
acids could be tolerated at this position; a Glu, Asn, or Gln
substitution did not result in a statistically significant difference
in CSF production. The latter finding indicates that the pre-
diction based on the consensus sequence that there is a hydro-
phobic residue at this position is not strictly correct. While
there is a preference for hydrophobic residues at the ?4 po-
sition, some polar residues can be tolerated.
Conclusions and implications. In this study, we identified
residues that are important for release of the CSF signaling
peptide of B. subtilis from its precursor protein, PhrC. We
previously identified a loose consensus sequence for five resi-
dues preceding the cleavage sites that produce the mature Phr
pentapeptides of B. subtilis (28) (Table 1). Analysis of substi-
tution of these five amino acid residues in PhrC, the precursor
protein for the Phr peptide CSF, revealed the importance of
these residues in directing cleavage of the precursor protein to
release CSF. However, the amino acid substitution data also
revealed that a relatively wide variety of sequences can be
tolerated at these positions and that this toleration may be due
to the fact that multiple proteases are able to cleave PhrC.
PhrC appears to tolerate a relatively wide variety of se-
quences in the residues preceding the cleavage site, resulting in
CSF production that is relatively robust to mutational pertur-
bation. For example, it was observed that only 4 of 19 amino
acid substitutions at the ?4 position resulted in a ?2-fold
defect in CSF production. This robustness to mutational per-
turbation appears to be due to the presence of multiple, re-
dundant proteases that process CSF. If subtilisin were the only
protease that processes PhrC to CSF, we would have observed
that a Val-to-Glu substitution at the ?2 position severely de-
creased CSF production. Instead, this substitution had a mod-
erate effect on CSF production because Vpr is able to process
PhrC with this substitution, when subtilisin cannot process it. It
is interesting that a Glu residue occurs at the ?2 position of
PhrE and PhrH (with a one-residue gap allowed for the PhrH
alignment, although it is difficult at this time to accurately
predict the sequence of the mature PhrH peptide). Given the
ability of Vpr to process a PhrC substrate with a Glu residue at
the ?2 position, we predict that Vpr and not subtilisin plays a
significant role in processing of the PhrE and PhrH peptides.
Further work could determine whether production of these
Phr peptides exhibits robustness to mutational perturbation
similar to that of CSF.
These studies contributed to our goal of identifying the
proteases that process Phr peptides of B. subtilis and other
bacteria. Even though CSF production was tolerant to many
amino acid substitutions, identifying substitutions such as the
Phe-to-Lys substitution at the ?4 position relative to the cleav-
age site for CSF should allow us to test whether production of
other Phr peptides is similarly disrupted by such a substitution.
Of course, one method to test whether subtilisin, Vpr, or Epr
has a role in cleaving other Phr peptides is to test these pro-
teases in vitro to determine whether they have this processing
activity, as we have done with PhrA (15). Showing that analo-
gous amino acid substitutions that affect processing of CSF by
subtilisin and Vpr in vitro also affect processing of a Phr
peptide, such as PhrA, would provide in vivo support for the
hypothesis that these Phr peptides are processed by the
same proteases. One question that is important to answer
before substitutional analyses of other Phr precursor pro-
teins are performed is whether there is flexibility in the
position of the residues that direct cleavage of Phr peptides.
As shown in Table 1, in order to obtain maximal alignment of
the Phr precursor proteins, it was necessary to introduce a
one-residue gap between the cleavage site and the consensus
sequence for a few of the Phr peptides. Future studies need to
address how this additional amino acid affects the residues
required for cleavage of the Phr precursor proteins. In the long
term, these studies have laid the foundation for determining
the mechanism of production of Phr peptides in B. subtilis and
FIG. 5. Amino acid substitutions at the ?4 position relative to the
cleavage site of PhrC. The Phe at position 32 of PhrC was replaced by
the other 19 canonical amino acids. The levels of endogenous CSF
production by cells expressing the mutant PhrC proteins were deter-
mined and normalized to the level of CSF production by the strain
expressing wild-type PhrC (amino acid F). The bars indicate the aver-
ages for at least three independent experiments, and the error bars
indicate the standard errors of the means. The asterisks indicate mu-
tant PhrC strains that were determined to be significantly different
from the wild-type strain by the Student t test (P ? 0.05).
6674 LANIGAN-GERDES ET AL.J. BACTERIOL.
This work was supported by NIH Public Health Service grant
AI48616 to B.A.L. S.L. was supported in part by USPHS National
Research Service award GM07104. The mass spectrometer used in this
work was purchased in part with funds from the W. M. Keck Founda-
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