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SpoIIE Is Necessary for Asymmetric Division, Sporulation, and Expression of

F, E, and G but Does Not Control Solvent Production in Clostridium
acetobutylicum ATCC 824

Article  in  Journal of Bacteriology · July 2011

DOI: 10.1128/JB.05474-11 · Source: PubMed

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JOURNAL OF BACTERIOLOGY, Oct. 2011, p. 5130–5137 Vol. 193, No. 19
0021-9193/11/$12.00 doi:10.1128/JB.05474-11
Copyright © 2011, American Society for Microbiology. All Rights Reserved.

SpoIIE Is Necessary for Asymmetric Division, Sporulation, and

Expression of ␴F, ␴E, and ␴G but Does Not Control Solvent
Production in Clostridium acetobutylicum ATCC 824䌤‡
Changhao Bi,1† Shawn W. Jones,1,2† Daniel R. Hess,3
Bryan P. Tracy,1,2,4 and Eleftherios T. Papoutsakis1*
Molecular Biotechnology Laboratory, Department of Chemical Engineering, and Delaware Biotechnology Institute, University of
Delaware, Newark, Delaware 197111; Department of Chemical and Biological Engineering, Northwestern University,
Evanston, Illinois 602082; Department of Chemistry and Biochemistry and Delaware Biotechnology Institute,
University of Delaware, Newark, Delaware 197113; and Elcriton Inc., 15 Innovation Way,
Room 288, Newark, Delaware 197114
Received 7 June 2011/Accepted 16 July 2011

In order to better characterize the initial stages of sporulation past Spo0A activation and the associated
solventogenesis in the important industrial and model organism Clostridium acetobutylicum, the spoIIE gene
was successfully disrupted and its expression was silenced. By silencing spoIIE, sporulation was blocked prior
to asymmetric division, and no mature spores or any distinguishable morphogenetic changes developed. Upon
plasmid-based complementation of spoIIE, sporulation was restored, although the number of spores formed
was below that of the plasmid control strain. To investigate the impact of silencing spoIIE on the regulation of
sporulation, transcript levels of sigF, sigE, and sigG were examined by semiquantitative reverse transcription-
PCR, and the corresponding ␴F, ␴E, and ␴G protein levels were determined by Western analysis. Expression
of sigF was significantly reduced in the inactivation strain, and this resulted in very low ␴F protein levels.
Expression of sigE was barely detected, and no sigG transcript was detected at all; consequently, no ␴E or ␴G
proteins were detected. These data suggest an autostimulatory role for ␴F in C. acetobutylicum, in contrast to
the model organism for endospore formation, Bacillus subtilis, and confirm that high-level expression of ␴F is
required for expression of ␴E and ␴G. Unlike the ␴F and ␴E inactivation strains, the SpoIIE inactivation strain
did not exhibit inoculum-dependent solvent formation and produced good levels of solvents from both expo-
nential- and stationary-phase inocula. Thus, we concluded that SpoIIE does not control solvent formation.

Clostridia are an ancient, diverse class and genus of anaer-
obic, endospore-forming bacteria, ranging from pathogenic to
solventogenic and cellulolytic species (34). The process of clos-
tridial endospore formation is still poorly understood, and this
has hindered progress in understanding their physiology and
their pathophysiological impact and devising strategies to en-
gineer strains for industrial applications (25, 33). Understand-
ing how the sporulation process is initiated, propagated, and
sustained is especially important in Clostridium acetobutylicum,
where both sporulation and solvent formation are transcrip-
tionally activated by Spo0A, the master transcription factor of
sporulation (14). Although C. acetobutylicum has not been
widely used industrially for its acetone, butanol, and ethanol
(ABE) fermentation for several decades (19), interest in this
and related organisms has intensified recently because of their
potential to produce not only butanol as a chemical and biofuel
(25, 33) but also butyrate as an industrial chemical and biofuel
precursor (33).
It is still broadly assumed that the regulation of sporulation
* Corresponding author. Mailing address: Delaware Biotechnology In-
stitute, University of Delaware, 15 Innovation Way, Newark, DE 19711.
Phone: (302) 831-8376. Fax: (302) 831-4841. E-mail: epaps@udel.edu.
† Joint first authors.
‡ Supplemental material for this article may be found at http://jb
.asm.org/.
䌤
Published ahead of print on 22 July 2011.
in clostridia is similar to that in the model organism for endo-
spore formation, Bacillus subtilis. In B. subtilis, sporulation is
regulated by a cascade of different transcription/sigma factors,
starting with Spo0A and followed by the prespore-specific
sigma factor ␴F and the mother cell-specific ␴E (16). In the
prespore, ␴F expression is followed by ␴G expression, and in
the mother cell, ␴E expression is followed by ␴K expression
(16). Expression and activation of each of these sigma factors
are tightly controlled and generally subject to both transcrip-
tional and posttranslational control (16). The gene sigF, en-
coding the first sigma factor to become active (␴F), is part of
a tricistronic operon with spoIIAA and spoIIAB (10, 36) and
is upregulated by phosphorylated Spo0A (Spo0A⬃P) (45,
46). Though translated, ␴F is kept inactive by being seques-
tered by SpoIIAB until after asymmetric division (37). Fol-
lowing asymmetric division, the anti-anti-sigma factor SpoI-
IAA is dephosphorylated by the membrane-bound
phosphatase SpoIIE and the dephosphorylated SpoIIAA
binds to SpoIIAB to release ␴F (2, 8, 23). In addition to its
role in activating ␴F, SpoIIE also plays a key role in asym-
metric division. SpoIIE has been found to directly interact
with FtsZ (6, 22, 26, 28), a tubulin-like protein, and this
interaction, along with an increase in FtsZ protein levels,
results in the repositioning of the FtsZ ring from the center
of the cell to a ring at each pole of the cell, where one of the
rings eventually forms the asymmetric septum (6, 9). It is
through these two roles, helping to position the asymmetric

5130
VOL. 193, 2011
FtsZ ring and releasing (and thus activating) ␴F from SpoI-
IAB, that SpoIIE plays an essential part in the sporulation
process of B. subtilis.
The C. acetobutylicum homolog of spoIIE has been identified
and annotated (31), and its expression has been confirmed (1,
20). Expression of spoIIE is initiated during early stationary
phase and reaches its peak shortly thereafter (20). This profile
generally matches with the promoter activity of spoIIE in B.
subtilis, where activity begins during early stationary phase and
continues into mid-stationary phase (11). However, when a
similar promoter activity study was performed on spoIIE in C.
acetobutylicum, activity was not noted until mid-stationary
phase, with a peak in expression soon after that (38). In order
to investigate the role of spoIIE in sporulation and solvento-
genesis, the gene was downregulated using antisense RNA
(38). Upon knockdown of spoIIE, solventogenesis in the strain
was slightly prolonged, and sporulation was significantly de-
layed compared to a plasmid control strain (38).
In B. subtilis, strains with mutations in spoIIE had similar
phenotypes to sigF and sigE mutants, in that sporulation was
blocked at stage II (asymmetric division) and that disporic cells
developed (17). The asymmetric septa that formed were thick
septal structures, like those generally seen during vegetative
growth, and not the thinner septal structures typically seen
during sporulation (5). In contrast to the B. subtilis sigF and
sigE mutants, sporulation was blocked prior to asymmetric
division in C. acetobutylicum mutants FKO1 and EKO1 with
silenced sigF (21) and sigE (43) genes, respectively. If SpoIIE
plays a similar role in sporulation in C. acetobutylicum as in B.
subtilis, a similar phenotype as the FKO1 mutant would be
expected, since ␴F would not become active. In order to test
this hypothesis, the spoIIE gene was inactivated and the result-
ing strain was characterized. We found that, like FKO1,
sporulation was blocked prior to asymmetric division and that
expression of ␴F, ␴E, and ␴G was severely reduced or elimi-
nated altogether. The inactivation strain was successfully com-
plemented using plasmid expression (a process that has proved
unsuccessful in the sigF, sigE, and sigG inactivation strains of C.
acetobutylicum [21, 43]) to reinstate sporulation and expression
of all three sigma factors. Solvent formation in both the spoIIE
inactivation strain and the complemented strain was also ex-
amined.
MATERIALS AND METHODS
Bacterial strains, growth, and maintenance conditions. All bacterial strains
used in this study are listed in Table S1 in the supplemental material, along with
their relevant properties. Strains were grown and maintained as described in
reference 43.
Analytical methods. Cell growth was monitored by measuring the optical A600
with a DU 730 spectrophotometer (Beckman-Coulter, Brea, CA). Culture su-
pernatant samples were analyzed for glucose, acetate, acetoin, ethanol, butyrate,
acetone, and butanol with an Agilent (Santa Clara, CA) high-performance liquid
chromatograph (HPLC) (41). DNA concentrations and purities were measured
with a NanoDrop (Wilmington, DE) spectrophotometer.
Bacterial transformations. A Gene Pulser Xcell electroporation system (Bio-
Rad, Hercules, CA) was used to transform both Escherichia coli and C. aceto-
butylicum. Prior to transformation into C. acetobutylicum, plasmids were meth-
ylated in E. coli carrying the methylating plasmid pAN2, a plasmid in which the
tetracycline and ampicillin resistance cassettes in pAN1 (29) were replaced with
a chloramphenicol resistance cassette, and clostridial transformations were per-
formed as described in reference 30.
Construction of disruption and complementation plasmids. The plasmids
used in this study are listed in Table S1 in the supplemental material, and the
INACTIVATION OF SpoIIE BLOCKS SPORULATION 5131
primers used are listed in Table S2 in the supplemental material. To disrupt the
spoIIE gene (CAC3205), the plasmid pKOSPOIIE was constructed. First, a
587-bp region of the spoIIE gene was PCR amplified with AmpliTaq Gold DNA
polymerase (Applied Biosystems, Foster City, CA) from C. acetobutylicum
genomic DNA using the primer set spoIIE-frag-F/R (see Table S2 in the sup-
plemental material), and the product was cloned into the pCR8/GW/TOPOTA
cloning plasmid (Invitrogen, Carlsbad, CA) and One Shot TOP10 E. coli (Invitro-
gen, Carlsbad, CA), following the manufacturer’s instructions. The resulting plasmid
is called pCR8-SPOIIE. This plasmid was then linearized with DraI (New England
BioLabs [NEB], Ipswich, MA), which has a single digestion site approximately in the
middle of the spoIIE fragment, and the ends were dephosphorylated with Antarctic
phosphatase (NEB, Ipswich, MA). A chloramphenicol/thiamphenicol resistance
(Cm/Thr) cassette optimized for C. acetobutylicum (39) was ligated into pCR8-
SPOIIE using a Quick Ligation kit (NEB, Ipswich, MA) and cloned into One Shot
TOP10 E. coli (Invitrogen, Carlsbad, CA), following the manufacturer’s instructions.
The resulting plasmid is called pCR8-SpoIIE/CM. The Cm/Thr cassette encodes
chloramphenicol resistance in E. coli and thiamphenicol resistance in C. acetobuty-
licum. Finally, the SPOIIE/CM gene disruption cassette was recombined into the
Destination vector pKOREC (43) using Invitrogen’s Gateway system (Carlsbad,
CA). The resulting plasmid is called pKOSPOIIE.
To complement the disruption strain, the plasmid pSPOIIE was constructed.
The entire spoIIE gene with its full upstream and downstream intergenic regions
was amplified from C. acetobutylicum genomic DNA using Phusion High-Fidelity
DNA polymerase (Finnymes Oy, Espoo, Finland) and the primer set spoIIE-F/R
(see Table S2 in the supplemental material). The PCR product was then cloned
into the pCR8/GW/TOPOTA cloning plasmid (Invitrogen, Carlsbad, CA) and
One Shot TOP10 E. coli (Invitrogen, Carlsbad, CA), following the manufactur-
er’s instructions. This plasmid, pCR8-FULL-SPOIIE, was then recombined into
the pDEST-TC Destination plasmid using the Invitrogen (Carlsbad, CA) Gate-
way LR recombination reaction to yield pSPOIIE. pDEST-TC was constructed
by first linearizing the tetracycline-based plasmid pTLH1 (13) with PvuII (NEB,
Ipswich, MA). This linearized plasmid was then dephosphorylated, ligated to a
phosphorylated Destination cassette from pcDNA-DEST40 (Invitrogen, Carls-
bad, CA), and cloned into DB3.1 ccdB resistant E. coli cells (Invitrogen, Carls-
bad, CA). Tetracycline was used only with solid agar plates because of the
negative effects on clostridial growth and metabolism in liquid media (12).
Inducing and confirming chromosomal integration. Chromosomal integration
of pKOSPOIIE was achieved using a similar protocol as described in reference
43. C. acetobutylicum pKOSPOIIE was grown to mid-exponential phase, and 150
␮l of culture was plated onto solid 2⫻ YTG plates (32) supplemented with 5
␮g/ml thiamphenicol (Th). After 24 h of growth, colonies were vegetatively
transferred onto fresh 2⫻ YTG plates with 5 ␮g/ml of thiamphenicol every 24 h
for a total of six transfers. Colonies were then plated onto fresh 2⫻ YTG plates
with no antibiotic and vegetatively transferred every 24 h for a total of four
transfers in order to cure the cells of the plasmid. Colonies were then transferred
onto 2⫻ YTG plates with 5 ␮g/ml of thiamphenicol and onto 2⫻ YTG plates
with 40 ␮g/ml of erythromycin, and putative integrants were determined as
described in reference 43. Though a double-crossover event was preferred, only
single-crossover events were found in the screening process, similar to past
studies using this protocol (21, 43). Finally, colony PCR (39) was used to deter-
mine through which region of homology integration was achieved.
Southern blot analysis. Genomic DNA from wild type (WT) and the spoIIE
mutant (SPOIIEKO) was isolated using Qiagen’s Genomic-tip 100/G system
(Valencia, CA), following the manufacturer’s instructions. Following digestion,
500 ng of genomic DNA and 1 ng of plasmid DNA were loaded onto the gel and
separated by gel electrophoresis. Blots were prepared, probed, and imaged as
described in reference 43. Probe template was generated from WT genomic
DNA using the primers SpoIIE-probe-F/R (see Table S2 in the supplemental
material) and Phusion High-Fidelity DNA polymerase (Finnymes Oy, Espoo,
Finland). Probes were then biotinylated using the NEBlot Phototope kit (NEB,
Ipswich, MA), following the manufacturer’s instruction.
RNA isolation and semiquantitative reverse transcription-PCR (RT-PCR).
Cells were sampled and RNA was isolated as described in reference 20. RNA
was reversed transcribed into cDNA as described in reference 7, except that 700
ng of total RNA was reverse transcribed. Primers were designed to amplify the
mature, full transcript and are listed in Table S2 in the supplemental material.
All products were amplified using AmpliTaq Gold DNA polymerase (Applied
Biosystems, Foster City, CA).
Western blot analysis. Crude cell extracts were prepared and total protein
concentration was determined as described in reference 43. For Western blots,
50 ␮g of total protein was loaded onto the gels, and blots were prepared and
imaged as described in reference 43. Primary antibodies (affinity-purified, poly-
clonal antibodies custom-made by ProteinTech [Chicago, IL] in rabbits) were
5132 BI ET AL. J. BACTERIOL.

FIG. 1. Disruption of the spoIIE gene. (A) The pKOSPOIIE vector and the spoIIE locus on the C. acetobutylicum ATCC 824 genome. The two
regions of homology are indicated on both the plasmid and the chromosome. (B) Integration of pKOSPOIIE into the genome. The plasmid
underwent a single crossover through the 2nd homologous region. The locations of the confirmation primers along with the promoter (Pptb) (44)
and rho-independent terminator (RIT) of the Cm/Thr cassette are indicated. (C) Schematic of how pKOSPOIIE would have integrated had a single
crossover through the 1st homologous region occurred. The locations of the confirmation primers are indicated. (D) PCR results for the
confirmation primers used on SPOIIEKO. The sizes demonstrate that pKOSPOIIE integrated by a single crossover through the 2nd region of
homology.

diluted to the following: Spo0A, 1:2,000; ␴F, 1:250; ␴E, 1:250; and ␴G, 1:250.
Secondary antibodies (goat anti-rabbit IgG, horseradish peroxidase conjugated
[Cell Signaling Technology, Danvers, MA]) were diluted to 1:3,000. All antibod-
ies were incubated with the membrane for 1 h at room temperature, except for
the antibody against ␴E, which was incubated overnight at 4°C.
Sporulation assays. Heat shock and chloroform spore assays were performed
as described in reference 43.
Microscopy. Phase-contrast microscopy samples were prepared as described in
reference 42 and were imaged on a Zeiss (Thornwood, NY) Axioskop 2 micro-
scope. Transmission electron microscopy (TEM) samples were prepared and
imaged as described in reference 20.
RESULTS
Integration of pKOSPOIIE to disrupt spoIIE. The phospha-
tase spoIIE (CAC3205) (31) was disrupted using a replicating
plasmid with two regions of homology (the 1st region being 294
bp long and the 2nd region being 293 bp long) (Fig. 1A),
similar to the approaches used to disrupt sigF (21) and sigE and
sigG (43). Though a double-crossover event was desired (to
obtain colonies resistant to thiamphenicol but sensitive to
erythromycin), only single-integration mutants (colonies resis-
tant to both thiamphenicol and erythromycin) were detected in
the screening, similar to previous attempts using comparable
plasmids (21, 43). In order to determine whether the single
integration occurred through the 1st or 2nd homologous re-
gion, the following five primers were used: spoIIE-KO-F,
spoIIE-KO-R, TH-F, PB-F, and PB-R (Fig. 1; see Table S2 in
the supplemental material). Putative integration colonies, from
here on referred to as SPOIIEKO, were selected for colony
PCR (39) using the primer sets PB-F/spoIIE-KO-R, TH-F/
spoIIE-KO-R, and spoIIE-KO-F/PB-R (Fig. 1). If integration
occurred through the 1st homologous region, the three PCR
primer sets would have produced bands of 998 bp, 6,308 bp,
and 2,840 bp, respectively (Fig. 1C), while if the integration
occurred through the 2nd homologous region, the products
would have been 2,046 bp, 1,088 bp, and 1,791 bp, respectively
(Fig. 1B). Following amplification, bands corresponding to in-
tegration through the 2nd region were produced (Fig. 1D). In
addition, the entire integration region was PCR amplified and
sequenced. The integration region could not be amplified as a
whole, but overlapping regions were amplified and sequenced
(data not shown). The sequencing results confirmed that a
single copy of pKOSPOIIE integrated through the 2nd homol-
ogous region.
Furthermore, genomic DNA from both WT and SPOIIEKO
C. acetobutylicum was prepared for Southern blotting to dem-
onstrate that pKOSPOIIE integrated only within the spoIIE
locus. Genomic DNA from both strains was digested with
VOL. 193, 2011 INACTIVATION OF SpoIIE BLOCKS SPORULATION 5133

FIG. 2. Southern blot confirmation of pKOSPOIIE integration site. (A) NdeI-digested genomic DNA from C. acetobutylicum WT and
SPOIIEKO hybridized with a probe for the 2nd homologous region. Lane 1, NdeI-digested SPOIIEKO DNA; lane 2, BamHI-digested
pKOSPOIIE; lane 3, NdeI-digested pKOSPOIIE; lane 4, NdeI-digested WT DNA; lane L, 2-log ladder from NEB. Membrane was exposed
for 2 min. Diagrams of expected bands from WT (B), SPOIIEKO (C), and pKOSPOIIE (D) are shown.

NdeI. This restriction enzyme has two digestion sites flanking

the spoIIE locus (Fig. 2B) and a single digestion site on the
plasmid backbone (Fig. 2D). After hybridization with the
probe, a single band (3.45 kb) should be produced from di-
gested WT genomic DNA, while two bands (6.4 kb and 3.3 kb)
should be produced from digested SPOIIEKO genomic DNA.
NdeI- and BamHI-digested pKOSPOIIE was also prepared
(Fig. 1D) and probed as positive controls and size markers.
The NdeI-digested plasmid is 6.3 kb, about the same size as the
upper band for SPOIIEKO genomic DNA, while the BamHI-
digested plasmid produces a 3.4-kb fragment, about the same
size as the lower SPOIIEKO band and the WT band. Only two
bands were detected for the disruption strain, and their sizes
were about what was expected (⬃6.4 kb and ⬃3.3 kb) (Fig.
1A). This confirms that pKOSPOIIE integrated only within the
spoIIE locus and not within any other loci on the chromosome.
Silencing of spoIIE significantly reduces the expression of
sigF and prevents the expression of sigE and sigG. In the
absence of an antibody against SpoIIE, semiquantitative RT-
PCR was used to prove that expression of spoIIE is silenced by
the integration of pKOSPOIIE. RNA was isolated from WT
and SPOIIEKO cultures after 48 h of growth (growth profiles
are displayed in Fig. S1 in the supplemental material) and
reverse transcribed into cDNA. Primers were designed to am- FIG. 3. Expression of spoIIE and sporulation-related trans-
plify a 1,812-bp fragment of the full transcript, which spans the criptional regulators in C. acetobutylicum WT, SPOIIEKO, and
site of disruption. Following amplification, a band was detected SPOIIEKO(pSPOIIE). (A) Semiquantitative RT-PCR of spoIIE,
spo0A, sigF, sigE, and sigG in WT, SPOIIEKO, and SPOIIEKO
for WT, but no bands were detected for SPOIIEKO (Fig. 3A). (pSPOIIE). Transcript expression was tested at 48 h after inocula-
A biological replicate produced the same results (data not tion. ⫹, reactions in which reverse transcriptase was added to the
shown). As a positive control, a 622-bp fragment of the full reaction mixture; ⫺, reactions in which reverse transcriptase was
spo0A transcript was also amplified and was detected in both not included in the reaction mixture. Products were imaged on an
ethidium bromide-stained 1% agarose gel. (B) Western blot anal-
WT and SPOIIEKO (Fig. 3A). ysis of Spo0A, ␴F, ␴E, and ␴G in WT, SPOIIEKO, and SPOIIEKO
Next, the expression of three of the major sporulation-re- (pSPOIIE). The ␴F image with an asterisk to the right was exposed
lated sigma factors, ␴F, ␴E, and ␴G, was investigated in WT for twice as long as the image above it.
5134 BI ET AL.
FIG. 4. Microscopy of C. acetobutylicum SPOIIEKO after 72 h of
growth. (A) Phase-contrast microscopy of SPOIIEKO. Only rod-
shaped cells with no identifiable differentiation phenotypes are evi-
dent. (B) TEM of SPOIIEKO. White, dashed arrows indicate the
developing symmetrical septal membrane.
and SPOIIEKO cultures. First, mRNA expression was exam-
ined using semiquantitative RT-PCR. A sigF band was de-
tected in SPOIIEKO, but its intensity was significantly lower
than that in WT, while a sigE band was barely detected
in SPOIIEKO (Fig. 3A). No sigG band was detected in
SPOIIEKO. Next, the protein expression of these same
three sigma factors was tested. Though a ␴F band was de-
tected, it was a very weak band, and no bands were detected
for either ␴E or ␴G (Fig. 3B). These results demonstrate that
silencing of spoIIE in SPOIIEKO affects the expression of
the sporulation-related sigma factors ␴F, ␴E, and ␴G.
Sporulation and all morphological changes are abolished in
SPOIIEKO. Previously, when the sigF or sigE genes were dis-
rupted and inactivated, the corresponding strains, FKO1 (21)
and EKO1 (43), respectively, showed very few signs of differ-
entiation and neither formed mature spores. For both strains,
transmission electron microscopy (TEM) images revealed
slight electron-translucent regions at one or both poles of the
cell (21, 43), and the EKO1 strain produced one or more
internal, longitudinal membranes (43). Therefore, with the
very weak expression of ␴F and the lack of ␴E (Fig. 3B), it was
not expected that SPOIIEKO would display many, if any, signs
of differentiation. Phase-contrast microscopy images after 72 h
of growth show only rod-shaped, exponential-phase-like cells
with no differentiation forms, such as clostridial or endospore
forms (18, 27, 42), detected (Fig. 4A). The cells were further
examined under TEM, and again, only rod-shaped cells with
no defining internal structures were found (Fig. 4B), in con-
trast to the FKO1 (21) and EKO1 (43) strains. Interestingly,
even though cells were within mid-stationary phase, some di-
viding cells with a septum developing in the middle of the cell
were still detected (Fig. 4B). However, no asymmetric division
was visible in any of the cells imaged. Finally, when late-
J. BACTERIOL.
FIG. 5. Sporulation of complemented strain SPOIIEKO(pSPOIIE).
The numbers of CFU of the plasmid control strain 824(pTLH1) and the
complemented strain SPOIIEKO(pSPOIIE) are shown. Standard devia-
tions between two biological replicates and two technical replicates (n ⫽
4) are indicated.
stationary-phase cultures (⬎5 days old) were submitted to ei-
ther chloroform treatment or heat treatment, no colonies sur-
vived, verifying that SPOIIEKO does not produce mature
spores. In contrast, WT cultures generally produce at least 1 ⫻
105 CFU/ml of spores by day 5.
Plasmid-based complementation of strain SPOIIEKO re-
stores sporulation. In order to complement the SPOIIEKO
strain, we constructed plasmid pSPOIIE. The spoIIE gene
along with the full upstream and downstream intergenic re-
gions was cloned into a tetracycline-based backbone. Presum-
ably, the upstream region includes the promoter and ribosomal
binding site for spoIIE and any other regulatory elements, and
the downstream region should contain a transcriptional termi-
nator, though no stem-loop structure was found downstream
of spoIIE (24). In the complemented strain, SPOIIEKO
(pSPOIIE), expression of spoIIE mRNA was restored to WT
levels, as were the mRNA and protein levels of all three of the
tested sigma factors, ␴F, ␴E, and ␴G (Fig. 3).
The strain was also able to produce mature spores, although
to a lesser extent than the plasmid control strain 824(pTLH1)
(Fig. 5). 824(pTLH1) produced over 1 ⫻ 104 CFU/ml of spores
by 96 h (4 days), while the complemented strain SPOIIEKO
(pSPOIIE) produced only about 5 ⫻ 102 CFU/ml by 120 h (5
days) (Fig. 5). Though production was significantly lower than
the plasmid control, SPOIIEKO(pSPOIIE) was still able to
produce mature spores, which the strain SPOIIEKO failed to
do. It is significant to note that previous attempts to comple-
ment sporulation-related genes in C. acetobutylicum (notably,
sigF, sigE, and sigG) with multicopy plasmids were not success-
ful (21, 43). Also, the plasmid control strain 824(pTLH1) itself
produces fewer spores than typical WT cultures, which pro-
duce at least 1 ⫻ 105 CFU/ml of spores by 120 h (5 days) of
growth (21). Previous plasmid control strains would generally
sporulate to a greater extent than WT (42), though it is un-
known why this occurs, but this particular plasmid control with
the tetracycline resistance gene has not been tested for sporu-
lation before.
VOL. 193, 2011
TABLE 1. Endpoint metabolite concentrations after 120 h of growth
Phase of
Strain Consumed
inoculum used Butyrate
glucose
WT Exponential (n ⫽ 6) 413.2 (⫾18.9) 11.9 (⫾1.8)
WT Stationary (n ⫽ 6) 403.0 (⫾9.8) 13.1 (⫾1.3)
SPOIIEKO Exponential (n ⫽ 6) 321.2 (⫾8.9) 24.0 (⫾5.2)
SPOIIEKO Stationary (n ⫽ 6) 311.8 (⫾23.4) 28.3 (⫾5.6)
pTLH1 Exponential (n ⫽ 2) 311.0 (⫾16.4) 18.8 (⫾2.1)
SPOIIEKO(pSPOIIE) Exponential (n ⫽ 2) 330.9 (⫾8.1) 16.3 (⫾4.6)
a
Values in parentheses show standard deviations.
Silencing of spoIIE does not abolish solvent production,
which remains independent of the inoculum age. When the
master sporulation transcription factor Spo0A was inactivated,
solvent production was virtually abolished because Spo0A di-
rectly upregulates a number of important solvent-formation
genes (14). When the early-acting sigma factors ␴F and ␴E
were inactivated (strains FKO1 and EKO1, respectively), sol-
vent production was found to be dependent upon the age of
the inoculum used (21, 43). When mid- to late-exponential
cultures were used as inocula, the levels of solvents produced
were significantly lower than typical WT levels, but when a
stationary-phase culture was used, solvent levels returned to
WT levels (21, 43). Since the phenotype of SPOIIEKO was
similar to the phenotypes of FKO1 and EKO1, a similar inoc-
ulum dependency in solvent formation was expected. However,
no inoculum dependency was found in SPOIIEKO (Table 1).
When either an exponential-phase or stationary-phase culture
was used for the inoculum, the cultures produced similar
amounts of butanol (⬃130 mM), acetone (⬃50 mM), and
ethanol (⬃20 mM), though these levels are slightly lower than
WT levels (Table 1). The SPOIIEKO strain produced higher
levels of acetoin and consumed smaller amounts of glucose,
possibly due to the premature culture termination due to ac-
cumulation of higher levels of butyrate. The complementation
strain SPOIIEKO(pSPOIIE) produced normal levels of sol-
vents without any inoculum-age dependency (Table 1). Over-
all, SpoIIE does not have a significant impact on solvent pro-
duction, although it appears to impact glucose utilization in
these simple batch cultures without pH control.
DISCUSSION
In this study, the role of SpoIIE in the sporulation process of
C. acetobutylicum was investigated by disrupting and inactivat-
ing its gene, spoIIE. In the inactivation strain SPOIIEKO,
expression of spoIIE was silenced, as expected, but surprisingly,
expression of sigF and its resulting protein, ␴F, was also signif-
icantly reduced (Fig. 3). In addition, very little sigE transcript
was detected, resulting in no protein detection, and no sigG
transcript or protein was detected (Fig. 3). These results fur-
ther support the finding from the sigF inactivation strain FKO1
that expression of ␴E and ␴G is dependent upon expression of
␴F (21). Although low levels of ␴F were detected, these appear
not to be sufficient to express ␴E or ␴G.
As stated, the decreased expression of sigF itself in SPOIIEKO
was not expected. In B. subtilis, SpoIIE plays two key roles in
sporulation. The first is during asymmetric division, where it
INACTIVATION OF SpoIIE BLOCKS SPORULATION 5135
Endpoint metabolite concn (mM)a
Acetate Acetoin Ethanol Acetone Butanol
0.5 (⫾0.8) 16.7 (⫾0.8) 29.8 (⫾3.0) 77.1 (⫾11.2) 181.6 (⫾7.9)
0.1 (⫾0.2) 16.6 (⫾0.8) 28.2 (⫾1.7) 86.4 (⫾12.4) 182.1 (⫾7.0)
1.9 (⫾1.9) 38.4 (⫾3.4) 20.4 (⫾1.0) 50.7 (⫾8.7) 130.4 (⫾9.9)
3.7 (⫾4.9) 36.2 (⫾5.3) 21.4 (⫾2.6) 50.8 (⫾4.9) 127.7 (⫾10.3)
0.0 (⫾0.0) 26.3 (⫾13.1) 25.5 (⫾0.1) 77.2 (⫾5.9) 165.1 (⫾6.7)
0.6 (⫾2.1) 27.4 (⫾10.4) 29.9 (⫾7.0) 82.0 (⫾13.4) 175.1 (⫾1.9)
interacts with FtsZ and plays a role in the repositioning of the
FtsZ ring around the poles of the cell (6, 9), and the second is
by activating ␴F by dephosphorylating SpoIIAA (2, 8, 23). In
neither of these roles should SpoIIE affect the expression of
the sigF transcript. After conducting a thorough literature
search, a study investigating the expression of sigF in a SpoIIE
inactivation B. subtilis mutant could not be identified. A pos-
sible explanation for the observation that inactivation of
SpoIIE affects the transcription of sigF is that, in C. acetobu-
tylicum, ␴F may stimulate its own expression. On the basis of
microarray analysis, it has been shown that sigF is upregulated
by Spo0A in C. acetobutylicum (40) and that sigF undergoes a
bimodal expression pattern (20). Our hypothesis is that
Spo0A⬃P initially upregulates the sigF operon, including the
regulators spoIIAA and spoIIAB, and the ␴F protein from this
initial expression of sigF is sequestered by SpoIIAB, like in the
B. subtilis model (37). Also like in the B. subtilis model, SpoIIE
then dephosphorylates SpoIIAA so that it can bind to SpoIIAB
and release ␴F (2, 8, 23). The released and active ␴F would
then go on to further upregulate the sigF operon, and this
would result in the bimodal shape of its expression pattern
(20). Without these higher levels of ␴F, expression of ␴E and
␴G is not possible, and without ␴E expression, sporulation is
blocked prior to asymmetric division, as in the sigE inactivation
strain (43). This hypothesis is further supported by the predic-
tion of a ␴F/G-binding motif (a motif recognized by both ␴F and
␴G) upstream of the sigF operon (35), though this has not been
experimentally validated.
Complementation of spoIIE restored sporulation in SPOIIEKO
(Fig. 5), but the level of sporulation was significantly lower
than that for the plasmid control. A possible explanation for
these lower levels, though doubtful, is polar effects due to the
disruption. CAC3204, the gene downstream of spoIIE, is an-
notated as a cell cycle protein (31), but it is not predicted to be
part of an operon with spoIIE (35). Instead, spoIIE in C.
acetobutylicum is predicted to be transcribed as a singleton and
not part of an operon (35), like spoIIE in B. subtilis (11). A
putative ␴A-binding motif was found upstream of both spoIIE
and CAC3204 (35), and these promoter regions were unaf-
fected by the disruption (Fig. 1B). A rho-independent termi-
nator was not found between spoIIE and CAC3204 (24), but
neither is one found downstream of the spoIIE gene in B.
subtilis (24). Finally, though the Pptb promoter of the Cm/Thr
cassette is a strong promoter, the rho-independent terminator
at the end of the cassette should prevent read-through into
CAC3204 (Fig. 1B).
5136 BI ET AL.
In contrast to both the sigF and sigE inactivation strains
FKO1 (21) and EKO1 (43), SPOIIEKO did not display an
inoculum dependency on its ability to produce solvents (Table
1). Though the solvent levels were slightly lower than WT
levels, the strain produced solvents when inoculated with ei-
ther exponentially growing cells or stationary-phase cells (Ta-
ble 1). So far, the only strains which display an inoculum
dependency on solvent formation are FKO1 and EKO1, and
the reason for this is unknown.
What remains unknown about the role of SpoIIE in sporu-
lation in C. acetobutylicum is its location within the cell and
what, if any, role it plays in asymmetric division. In B. subtilis,
SpoIIE is a membrane-bound enzyme and is located within the
septal membrane (3, 4). However, if SpoIIE must activate ␴F
prior to asymmetric division in C. acetobutylicum, it is unknown
where it would be located within the cell. If the hypothesis that
SpoIIE is active prior to asymmetric division is true, this ac-
tivity would have a profound impact on the C. acetobutylicum
model for sporulation. Instead of being a prespore-specific sigma
factor, as in B. subtilis, ␴F could be active throughout the predi-
visional cell and may have roles beyond just sporulation. Because
this hypothesis is in stark contrast to the B. subtilis model, more
experimental evidence is needed to support it, but as has been
seen in C. perfringens (15), sporulation in clostridia may be a
substantially different process than sporulation in bacilli.
ACKNOWLEDGMENTS
We acknowledge the use of the Delaware Biotechnology Institute
Bio-Imaging Facility for all microscopy images, and we thank Shannon
Modla for preparing and imaging the electron microscopy images.
This work was supported by National Science Foundation (NSF)
grant CBET-0853490.
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