�K of Clostridium acetobutylicum Is the First Known Sporulation-Specific Sigma Factor with Two Developmentally Separated Roles, OneEarly and One Late in Sporulation

Mohab A. Al-Hinai,a,c,d Shawn W. Jones,e Eleftherios T. Papoutsakisa,b,c,e

‹Department of Biological Sciences, University of Delaware, Newark, Delaware, USAa; Department of Chemical & Biomolecular Engineering, University of Delaware, Newark,Delaware, USAb; Molecular Biotechnology Laboratory, Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, USAc; Department of Biology, SultanQaboos University, Muscat, Omand; Elcriton Inc., New Castle, Delaware, USAe

Sporulation in the model endospore-forming organism Bacillus subtilis proceeds via the sequential and stage-specific activationof the sporulation-specific sigma factors, �H (early), �F, �E, �G, and �K (late). Here we show that the Clostridium acetobutyli-cum �K acts both early, prior to Spo0A expression, and late, past �G activation, thus departing from the B. subtilis model. The C.acetobutylicum sigK deletion (�sigK) mutant was unable to sporulate, and solventogenesis, the characteristic stationary-phasephenomenon for this organism, was severely diminished. Transmission electron microscopy demonstrated that the �sigK mu-tant does not develop an asymmetric septum and produces no granulose. Complementation of sigK restored sporulation andsolventogenesis to wild-type levels. Spo0A and �G proteins were not detectable by Western analysis, while �F protein levels weresignificantly reduced in the �sigK mutant. spo0A, sigF, sigE, sigG, spoIIE, and adhE1 transcript levels were all downregulated inthe �sigK mutant, while those of the sigH transcript were unaffected during the exponential and transitional phases of culture.These data show that �K is necessary for sporulation prior to spo0A expression. Plasmid-based expression of spo0A in the �sigKmutant from a nonnative promoter restored solventogenesis and the production of Spo0A, �F, �E, and �G, but not sporulation,which was blocked past the �G stage of development, thus demonstrating that �K is also necessary in late sporulation. sigK isexpressed very early at low levels in exponential phase but is strongly upregulated during the middle to late stationary phase.This is the first sporulation-specific sigma factor shown to have two developmentally separated roles.

Clostridium acetobutylicum is a Gram-positive obligate anaer-obe that is capable of forming environmentally resistant en-

dospores (1). C. acetobutylicum has emerged as the model organ-ism in Clostridium genetics for investigating fundamental aspectsof growth, sporulation, metabolism, and product formation. Al-though C. acetobutylicum has been extensively studied due to itsability to produce commercially valuable solvents (2–4), detailedstudies on molecular aspects of its sporulation are quite recent(5–9). The sporulation model of Bacillus subtilis, the model organ-ism for studying sporulation in endospore formers, has long beenthought to be preserved in other Bacillus and Clostridium organ-isms (10). Although the major sporulation-related genes presentin B. subtilis, notably those of the master transcriptional regulatorof sporulation in all endospore formers, Spo0A, and the sporula-tion-specific sigma factors (�H, �F, �E, �G, and �K), have recog-nizable orthologs encoded by Clostridium genomes (11), evidenceis accumulating that regulation differs substantially betweenBacillus and Clostridium (6–8, 12, 13).

Spo0A has long been established as the switch that governs theinitiation of sporulation and solventogenesis, the characteristicstationary-phase phenomenon, in C. acetobutylicum and othersolventogenic Clostridium organisms (14–16). In B. subtilis, spo0Ais transcribed from a �H-dependent promoter (17). Once trans-lated, Spo0A is phosphorylated by a multicomponent phosphore-lay system to produce its active form (Spo0A�P) (10). In B. sub-tilis, Spo0A�P and �H positively regulate the expression of thespoIIA operon, which includes the gene that codes for the pres-pore-specific �F, the first of the four downstream sigma factors(10, 18). This results in the sequential, and stage-specific, produc-tion of the remaining sporulation-specific sigma factors down-

stream of �F (19, 20), namely, �E, �G, and �K. Spo0A�P simulta-neously induces sigE expression, the mother cell-specific sigmafactor (10, 21). Production of �G then follows in the prespore,while �K, the last sporulation-specific sigma factor, is produced inthe mother cell (10, 21). �K is necessary for driving the transcrip-tion of mother cell-specific genes, which include spore coat, sporecortex, and germination genes, whose products are necessary forthe successful conclusion of the sporulation cascade and the pro-duction of viable spores (21).

This sporulation cascade is substantially different in Clostrid-ium organisms. Inactivation of �F in C. acetobutylicum (8) abol-ished sporulation prior to stage II (asymmetric division). In con-trast, the B. subtilis sigF mutant did not enter stage III, and the cellswere disporic and appeared to have a normal septum (22). Fur-thermore, transcriptional analysis revealed that canonical genesunder the control of �F in B. subtilis did not show differentialexpression in the C. acetobutylicum sigF mutant, thus suggestingthat they are not under its transcriptional control (8). Unlike theB. subtilis sigE disruption mutant (22), for which the cells form anormal sporulation septum and exhibit a disporic phenotype, dis-

Received 16 September 2013 Accepted 23 October 2013

Published ahead of print 1 November 2013

Address correspondence to Eleftherios T. Papoutsakis, epaps@udel.edu.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.01103-13.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JB.01103-13

January 2014 Volume 196 Number 2 Journal of Bacteriology p. 287–299 jb.asm.org 287

on April 29, 2016 by guest

http://jb.asm.org/

Dow

nloaded from

ruption of sigE in C. acetobutylicum blocked sporulation prior toasymmetric division, and no disporic cells or granulose (a charac-teristic storage biopolymer of sporulating clostridia) accumula-tion was observed (6). On the other hand, the Clostridium perfrin-gens sigE disruption mutant was able to form a disporicphenotype, which is typical of sigE disruption mutants in B. sub-tilis, and the cells appeared to accumulate granulose but neverdeveloped beyond stage II (6, 12, 22). SpoIIID protein (a DNAbinding protein that aids in the transcriptional activation of thesigK gene in B. subtilis [23]) could be detected in the sigE disrup-tion mutant of C. perfringens, thus indicating that spoIIID is notunder �E transcriptional control, in contrast to the B. subtilismodel. Transmission electron microscopy (TEM) images revealedthat the C. acetobutylicum sigG disruption mutant had arrestedsporulation, and differentiation appeared to be blocked betweenstages III and IV, in contrast to sigG mutants of B. subtilis, in whichsporulation was blocked at stage III (6, 24, 25). It is unknown atwhat stage of sporulation the sigG disruption mutant of C. perfrin-gens was arrested since no TEM images were presented (26).

Disruption of sigK in C. perfringens resulted in abolished spo-rulation, and TEM images showed that only a small percentage ofcells developed any kind of asymmetric septa or accumulatedgranulose, thus suggesting that sporulation was blocked prior tostage II, which is earlier than in the C. perfringens sigE disruptionmutant (12). This is in contrast to findings for the B. subtilis sigKdisruption mutant, in which sporulation was blocked at stage IV(27). The �E protein in the C. perfringens sigK disruption mutantcould not be detected by Western analysis, thus confirming that�K is active upstream of �E in the sporulation cascade (12). A sigKdisruption mutant was also generated in Clostridium botulinum,and its sporulation was also blocked at an early stage (13). Quan-titative reverse transcription-PCR (qRT-PCR) analysis on thewild-type (WT) C. botulinum strain revealed that sigK expressionwas downregulated during early stationary phase. Moreover,spo0A and sigF transcripts were downregulated in the sigK disrup-tion mutant, and the authors concluded that �K is involved inearly stages of sporulation in C. botulinum (13). Most recently(28), sigK disruption in Clostridium difficile resulted in a largelyasporogenous phenotype, whereby forespores were surroundedby a layer that resembled the cortex layer of the wild type. In theirproposed models (28), there is no role for �K in early sporulationfor C. difficile. Most recently (29), C. difficile has been reclassifiedas belonging in the family Peptostreptococcaceae, with a new sug-gested name of Peptoclostridium difficile. Thus, the findings in ref-erence 28 may not reflect the regulation of sporulation in typicalClostridium organisms. Nevertheless, these authors (28) also pro-pose that in C. perfringens, �K is under the control of �F andnecessary for the production of pre-�E.

Here, we demonstrate that not only is �K necessary during theearly stages of sporulation in C. acetobutylicum but also that it isnecessary late to complete sporulation. Moreover, we aimed todetermine the precise points at which �K acts in early and, sepa-rately, late-stage sporulation. By expressing spo0A in the sigK de-letion mutant to bypass the early step in sporulation where �K isrequired, we were able to accurately assess its role during the latestages of sporulation and demonstrate its necessity for spore de-velopment. We show that sigK is expressed shortly after sporegermination, is expressed very early during exponential growth,and subsequently displays much higher expression in the middleto late stationary phase. These findings are in stark contrast not

only to the B. subtilis model but also to the recently proposedmodels for both C. difficile (P. difficile) and C. perfringens (28).Significantly, this is the first report of a sporulation-specific sigmafactor in an endospore former that is shown to have two develop-mentally separated roles, one very early and one very late in spo-rulation. We discuss several possible ramifications of this novelfinding.

MATERIALS AND METHODSBacterial strains and plasmids. Relevant characteristics of bacterialstrains and plasmids used in this work are listed in Table 1.

Construction of plasmids. All primers are listed in Table S1 in thesupplemental material. Plasmids pKOSIGK2_mazF and p94FLP_asRepLwere constructed as described previously (30). The pKISIGK_mazF com-plementation plasmid was designed to integrate the complete up- anddownstream regions of the sigK gene, including the open reading frame(ORF). Plasmid pKISIGK_mazF was constructed by digesting thepKOSIGK2_mazF plasmid with NotI (New England BioLabs [NEB],Ipswich, MA). The complete sigK gene, including the natural promoterand terminator, was PCR amplified from C. acetobutylicum genomic DNA(gDNA) with primers that incorporated NotI sites. The PCR product wasthen digested with NotI and ligated into the NotI (NEB)-linearizedpKOSIGK2_mazF, yielding plasmid pKISIGK_mazF. Accordingly, thepKISIGK_mazF plasmid was designed to reintegrate the sigK gene back inits natural genomic locus. The p94Spo0A plasmid was constructed bydoubly digesting the pSOS94 vector (31) with BamHI and KasI (NEB) andgel purifying the larger fragment. Subsequently, the spo0A gene was am-plified with primers that incorporated the BamHI and KasI sites and li-gated to the linearized vector, yielding plasmid p94Spo0A.

Culture conditions. Escherichia coli strains were grown aerobically at37°C and 220 rpm in liquid LB medium or solid LB medium with agar(1.5%) supplemented with 50 �g/ml of ampicillin (Amp) or 35 �g/ml ofchloramphenicol (Cm) as needed. Single C. acetobutylicum coloniesgrown anaerobically on solid 2� YTG medium (16 g/liter Bacto tryptone,10 g/liter yeast extract, 4 g/liter NaCl, and 5 g/liter glucose; pH 5.8) forat least 5 days were transferred to 10 ml of liquid clostridial growthmedium [CGM; 0.75 g/liter K2HPO4, 0.7 g/liter MgSO4·7H2O, 0.017g/liter MnSO4·5H2O, 0.01 g/liter FeSO4·7H2O, 2 g/liter (NH4)2SO4, 1g/liter NaCl, 2 g/liter asparagine, 0.004 g/liter p-aminobenzoic acid,5 g/liter yeast extract, 4.08 g/liter CH3COONa·3H2O, and 80 g/literglucose] (30) and heat shocked at 80°C for 10 min. Recombinantstrains were grown as before in media supplemented with 40 �g/ml oferythromycin (Em) for solid media and 100 �g/ml of Em for liquidmedia or 5 �g/ml of thiamphenicol (Th) as necessary. Where needed,40 mM �-lactose (Sigma-Aldrich, St. Louis, MO) was added to culturemedia to induce the expression from a lactose-inducible promoter.

Plasmid transformation. Newly constructed plasmids were trans-formed into E. coli Top10 (Life Technologies [LT], Grand Island, NY)cells and subsequently isolated and confirmed using restriction digests.Plasmids destined to be electrotransformed into C. acetobutylicum werefirst transformed into electrocompetent E. coli ER2275(pAN3) cells forDNA methylation. Plasmid pAN3 is a derivative of plasmid pAN1 exceptthat it has kanamycin resistance (32). Vectors were isolated and confirmedfrom their respective E. coli ER2275(pAN3) strains and subsequentlytransformed into C. acetobutylicum by electroporation in an anaerobicchamber as described previously (33), except that 5 to 10 �g of plasmidDNA was used to transform the cells.

Isolation of sigK double-crossover knockout mutants. Marked andunmarked (i.e., with the subsequent removal of the antibiotic resistancemarker via the flippase [FLP]-flippase recognition sequence [FRT] sys-tem) allelic-exchange sigK mutants were isolated as described previously(30).

Complementation of sigK mutants via chromosomal integration ofthe sigK gene. The sigK gene was integrated into its natural chromosomallocus. The pKISIGK_mazF plasmid was transformed into the �sigK_um

Al-Hinai et al.

288 jb.asm.org Journal of Bacteriology

on April 29, 2016 by guest

http://jb.asm.org/

Dow

nloaded from

strain, thiamphenicol resistance colonies were isolated, and the presenceof the plasmid was confirmed. Subsequently, chromosomal integrationmutants of the sigK gene were isolated by utilizing the vegetative liquidtransfer method previously described (30). The sigK complementationstrain was named SigK_comp.

Bioreactor experiments. Duplicate fermentations were carried out ina BioFlo 110 (New Brunswick Scientific Co., Edison, NJ) bioreactor witha 4.0-liter working volume as described previously (34). Culture pH wasmonitored in real time and maintained at or above 5.0 by actively adding6 N NH4OH to offset the drop in pH due to the buildup of the organicacids acetate and butyrate. All experiments were carried out in biologicalreplicates.

RNA isolation, sqRT-PCR, and qRT-PCR. RNA isolation, cDNA gen-eration, and semiquantitative reverse transcription-PCR (sqRT-PCR)were carried out as described previously (7), except that 2 �g of total RNAwas reverse transcribed. All amplification reactions were carried out usingPhusion high-fidelity DNA polymerase (NEB). qRT-PCR experimentswere carried out as described previously (35) with primers listed in TableS1 in the supplemental material and ca_c3571 as the housekeeping gene.To calculate the gene expression fold difference, using qRT-PCR analysis,between the �sigK and WT strains, RNA was extracted and cDNA wasgenerated at 6 h, 12 h, 20 h, and 26 h from pH-controlled bioreactorexperiments as described above. For the sigK time course gene expressionusing qRT-PCR analysis, WT cultures were grown in static flasks with a0.4-liter working volume. Subsequently, RNA extraction and cDNA gen-eration were done at 6 h, 15 h, 24 h, 30 h, 36 h, and 48 h. For sqRT-PCRexperiments, RNA extraction and cDNA generation for the �sigK andWT strains were done from cultures grown in pH-controlled bioreac-tors and from static flask cultures with a 0.4-liter working volume forthe SigK_comp, �sigK p94Spo0A, and EKO1 strains. All experimentswere conducted with two biological replicates.

Western blot analysis. Western blot analysis was carried out as de-scribed previously (6, 7) with 30 �g of total protein loaded onto the gels(except for WT Spo0A, for which only 15 �g was loaded, while only 5 �gwas loaded for the �sigK p94Spo0A strain).

Spore assays. Spore assays were carried out as described previously (6)from 5-day-old cultures obtained from the bioreactor experiments de-

scribed above for both the �sigK and WT strains. The SigK_comp and the�sigK p94Spo0A strains were grown in a 0.4-liter-working-volume staticflask culture. All experiments were done in two biological replicates.

Microscopy. Samples for transmission electron microscopy (TEM)were prepared and TEM imaging was carried out as described previously(9) from 5-day-old cultures.

Spore staining. The WT, �sigK, SigK_comp, and �sigK p94Spo0Astrains were streaked on 2� YTG plates (pH 5.8) and allowed to grow for5 days. Subsequently, a smear from each strain was made on a glass slideand stained with malachite green and safranin to visualize the spores (36).

Other analytical techniques. Cell density was measured at A600 with aBeckman Coulter DU 730 spectrophotometer using a 1-cm-gap cuvette.An Agilent (Santa Clara, CA) high-performance liquid chromatograph(HPLC) was used to analyze culture supernatant for glucose, acetoin,acetate, ethanol, butyrate, acetone, and butanol (37).

Confirmation of chromosomal insertions by PCR and sequencing.To confirm allelic-exchange chromosomal integration mutants, genomicDNA (gDNA) was isolated from cells using the DNeasy blood and tissuekit (Qiagen, Valencia, CA) by following the manufacturer’s protocol. Ap-propriate primer combinations (see Table S1 in the supplemental mate-rial) were used to confirm the integrants. Amplification reactions werecarried out using Phusion high-fidelity DNA polymerase (NEB). Mutantswere also confirmed by Sanger sequencing.

Microarray analysis. Genome-wide transcription analysis of the sigGmutant (GKO1) strain relative to the WT strain were conducted duringstationary phase, which corresponds to the peak expression of sigG in WTcells (9). Custom-built oligonucleotide Agilent Technologies 4x44k mi-croarrays (GEO accession number GPL10908) were hybridized, washed,and scanned according to the manufacturer’s instructions. The design ofthe probes is described in reference 38. Slide normalization (using theLOESS [locally weighted scatterplot smoothing] method) and averagingwere carried out using the Bioconductor package in R (39, 40). As de-scribed previously (9, 38), a minimum intensity of 50 intensity units wasused.

Microarray data accession number. Microarray data have been de-posited in the GEO database under accession number GSE24103.

TABLE 1 Bacterial strains and plasmids used in this study

Strain or plasmid Relevant characteristicsa

Source orreference

Strain name or mutationE. coli

ER2275 hsdR mcrA recA1 endA1 NEBTop 10 hsdR mcrA recA1 endA1 Life Technologies

C. acetobutylicumATCC 824 WT ATCC�sigK ATCC 824; mostly deleted sigK; Thr 30�sigK_um ATCC 824; unmarked sigK deletion 30SigK_comp �sigK_um; sigK complementation strain; Thr This study�sigK p94Spo0A �sigK p94Spo0A Thr MLSr This studyWT p94Spo0A WT p94Spo0A This study

PlasmidspAN3 Kmr; �3T I gene 30P94FLP_asRepL Emr; flippase; ptb promoter; bgaR and PbgaL upstream of asRNA to the repL origin 30pKOSIGK2_mazF Thr-FRT; MLSr; repL; ori; bgaR and PbgaL upstream of mazF; Thr-FRT flanked by

sigK �1,000 bp of up- and downstream region of homology30

pKISIGK_mazF pKOSIGK2_mazF; sigK with natural promoter and terminator This studyp94Spo0A spo0A; ptb promoter; MLSr This study

a hsdR, host-specific restriction deficient; mcrA, methylcytosine-specific restriction deleted; recA1, homologous recombination deleted; endA1, endonuclease deleted; ptb,phosphotransbutyrylase gene; Thr, thiamphenicol resistance; Kmr, kanamycin resistance; Thr-FRT, FRT-flanked thiamphenicol resistance; �3T I gene, B. subtilis phage �3T Imethyltransferase gene; MLSr, macrolide-lincosamide-streptogramin B resistance; mazF, codon-optimized E. coli MazF toxin gene; repL, pIM13 g-positive origin of replication; ori,ColE1 origin of replication; asRNA, antisense RNA.

�K of Clostridium acetobutylicum Plays Dual Roles

January 2014 Volume 196 Number 2 jb.asm.org 289

on April 29, 2016 by guest

http://jb.asm.org/

Dow

nloaded from

RESULTSInactivation of sigK abolishes sporulation early, prior to asym-metric division (stage II), and suppresses solventogenesis. Aknockout (KO) plasmid was designed to delete 664 bp of the705-bp sigK gene using two large noncontiguous regions of ho-mology (�1 kb) (Fig. 1A) that are up- and downstream of sigK asdescribed previously (30), and the strain was named the �sigKmutant (Fig. 1B). Unlike the sigK disruption mutants that weregenerated in C. perfringens (12) and C. botulinum (13), we wereunable to complement our sigK deletion mutant via plasmid ex-pression. We constructed three different plasmids each placingsigK expression under the control of its natural promoter, the ptbpromoter, or a lactose-inducible promoter. However, none of theplasmids were successful at complementing the �sigK strain. Thiswas not completely unexpected, since plasmid-based complemen-tation of the sigF, sigE, and sigG disruption mutants in C. acetobu-tylicum was also unsuccessful (6, 8). However, it illustrates thathigh levels of expression from a multicopy (albeit low-copy-num-ber) plasmid are detrimental to the cell and unable to restore theWT phenotype. Thus, we integrated the sigK gene, including thecomplete up- and downstream regions, back in the �sigK_umstrain (unmarked strain) (Fig. 1C) and named this mutant theSigK_comp strain (Fig. 1D). All strains were confirmed using PCR(Fig. 1E) and sequencing. The silencing of sigK and its restoredexpression in the complementation strain were confirmed withsqRT-PCR (Fig. 1F).

In order to ascertain the role of �K in the sporulation cascade,chloroform spore assays were employed to examine the �sigKstrain. Typically, WT cultures produce �1 �105 to 1 � 106

spores/ml (6, 8) after 5 days (120 h) in culture (Fig. 2A). The �sigKstrain failed to produce any spores during this time period (Fig.2A). On the other hand, the SigK_comp strain (the sigK comple-mentation strain) was able to restore sporulation to a level similarto that of the WT strain (Fig. 2A). Viable-cell counts, i.e., withoutchloroform treatment, for both the WT and SigK_comp strainswere ca. 1 � 106 CFU/ml, but for the �sigK strain they were be-tween 103 and 104 CFU/ml (Fig. 2A) after 5 days. Consistent withthe spore assays, spore staining showed that both the WT and SigK-_comp strains were able to produce spores, while the �sigK strain didnot develop any spores (see Fig. S1 in the supplemental material).

To examine morphological sporulation-related changes in�sigK, TEM images (Fig. 2B) were obtained for cells collectedfrom 5-day-old cultures. Typically, in the WT strain granulosevesicles and forespores can be readily identified (Fig. 2B). How-ever, in the �sigK strain no granulose, septum formation, or fore-spore structures could be identified (Fig. 2B). The TEM imagessuggest that the �sigK strain has sporulation blocked at an earlierstage, prior to stage II (asymmetric division), since no septa,granulose vesicles, or forespores could be identified. In contrast tothe C. perfringens sigK disruption mutant (12), for which a smallnumber of cells developed an asymmetric septum or showed adisporic phenotype or engulfment, none of many �sigK cells ex-amined exhibited any of those characteristics. As discussed above,in C. difficile (P. difficile) (28), sigK disruption resulted in a largelyasporogenous phenotype, whereby forespores were surroundedby a layer that resembled the cortex layer of wild-type cells. We areunable to assess the phenotype of the sigK disruption mutant gen-erated in C. botulinum (13) since no TEM images were presented,

FIG 1 Deletion, complementation, and confirmation of double-crossover allelic-exchange mutations at the sigK locus. (A) Wild type; (B) �sigK; (C) �sigK_um;(D) SigK_comp. The dashed lines in panel A show the approximate regions of homology (�1 kb each) incorporated in the knockout vector. (E) PCRconfirmation of double-crossover sigK deletion and complementation mutants with primers that anneal to the chromosome as indicated. (F) (sqRT-PCR) toconfirm the silencing of sigK expression in �sigK and the restored expression in the SigK_comp mutant. The WT is also shown. All experiments were done inbiological replicates (a and b). MW, HindIII digest ladder (NEB); M, 2-log ladder (NEB).

Al-Hinai et al.

290 jb.asm.org Journal of Bacteriology

on April 29, 2016 by guest

http://jb.asm.org/

Dow

nloaded from

but spore staining revealed that it did not develop spores and thecells appeared longer than normal (13). We conclude that sigKdeletion blocks sporulation early, prior to asymmetric division,with no evidence of septation or a disporic phenotype that wasobserved in the C. perfringens sigK disruption mutant (12).

Investigating sporulation in C. acetobutylicum has the advan-tage of the characteristic stationary-phase phenomenon of solven-togenesis (production of butanol, acetone, and ethanol), which is

regulated largely by an activated Spo0A protein (15) but is notabolished by the inactivation of any of the downstream (11) spo-rulation-specific factors, SpoIIE, �F, �E, and �G (6–8). Initial ex-periments with the �sigK strain using typical batch cultures with-out active pH control resulted in premature cell death due toexcessive medium acidification (“acid crash” [41]) from the pro-duction of butyric and acetic acids. WT C. acetobutylicum culturesproduce acetate and butyrate during exponential growth for gen-erating ATP for growth. In the transition and stationary phases,cells reassimilate the acids and produce acetone, butanol, and eth-anol. Acid crash indicates the inability of the cells to reassimilatethe acids. Thus, to assess the metabolic capabilities of the �sigKstrain, fermentation experiments with active pH control (pHmaintained above 5.0) were carried out. In these fermentor exper-iments, samples were taken after 120 h of culture and analyzed formetabolite concentrations (Table 2). WT cultures consumedmore than 370 mM glucose and produced 78 mM, 161 mM, and21 mM acetone, butanol, and ethanol, respectively (Table 2). Incontrast, �sigK cultures consumed 280 mM glucose and produced12 mM butanol and 2 mM (each) ethanol and acetone (Table 2).WT cultures produced 104 mM acetate and 39 mM butyrate, andthe �sigK cultures produced 130 mM acetate and 166 mMbutyrate (Table 2). These data show that the �sigK strain, in con-trast to the WT strain, cannot effectively reassimilate the organicacids that have accumulated in the culture during exponentialgrowth and cannot produce normal levels of solvents. This prod-uct formation profile of the �sigK strain is similar to that of theSpo0A inactivation strain SK01 (15).

Once the �sigK mutant was successfully complemented(SigK_comp strain), the ability to grow without active pH con-trol, as in the WT strain, was restored. SigK_comp cells con-sumed large amounts of glucose and produced solvent levelssimilar to those produced by the WT strain (Table 2).

Taken together, these data suggest that sigK deletion leads toabolished or suppressed production or activation of Spo0A, whichcontrols the expression of the downstream sporulation-specificfactors (spoIIE, sigF, sigE, or sigG) (6–8) as well as of the sol operon(adhE1[aad]-ctfAB), which encodes the key enzymes needed for ef-fective solventogenesis (15). To confirm these hypotheses, we usedWestern and qRT-PCR analyses as described immediately below.

Western blot analysis of the �sigK strain shows no detectablelevels of Spo0A or �G protein and severe downregulation of the�F protein; sigK complementation restores their production tonormal levels. Protein production of Spo0A, �F, and �G in the

FIG 2 The �sigK strain is unable to sporulate after 5 days. (A) Spore andviable-cell counts for the �sigK, SigK_comp, �sigK p94Spo0A, WT p94Spo0A,and WT strains after 5 days in culture. The �sigK and �sigK p94Spo0A strainswere unable to sporulate, while the SigK_comp strain sporulated successfully.All experiments were done in two biological replicates. Error bars representSEMs. The horizontal dashed line shows the lower detection limit of the assay.(B) TEM images of �sigK and WT strains. No granulose, septum, or foresporestructures were identified for the �sigK mutant, while the WT strain clearlyshows the development of a forespore (I), granulose vesicles (II), and a freespore (III). It is worth noting that the translucent mass on one pole of the �sigKstrain was also observed in the sigF mutant (FKO1), and it was originallyhypothesized to be condensed DNA. However, 4=,6-diamidino-2-phenylin-dole (DAPI) staining of the FKO1 strain showed a lack of DNA in that region(8). Thus, the composition of that mass remains unknown.

TABLE 2 Final metabolite concentrations at 120 h of culture

Culture type

Final product titer (mM)a

Consumed glucose Acetate Acetoin Ethanol Butyrate Acetone Butanol

BioreactorWT 376 (17.8) 104 (3.6) 11 (0.5) 21 (0.3) 39 (0.5) 78 (2.0) 161 (3.4)�sigK 282 (2.8) 130 (2.5) 12 (0.9) 2 (0.1) 166 (0.7) 2 (0.7) 12 (0.6)

Static flaskWT 312 (4.5) 1 (0.5) 9 (0.3) 29 (1.9) 13 (0.3) 91 (1.3) 162 (1.5)SigK_comp 325 (7.7) 2 (1.7) 23 (3.2) 51 (5.6) 23 (2.5) 72 (0.1) 159 (7.6)�sigK p94Spo0A 265 (37) 1.4 (1.2) 9.3 (1.7) 21.5 (3) 12 (0.3) 85 (7.8) 144 (11.5)WT p94Spo0A 216 (44) 7 (7) 7.4 (3) 17 (4) 26 ( 11) 49 (20) 91 (35)�sigK — — — — — — —

a Data are averages of two biological replicates. Values in parentheses represent standard deviations. —, culture did not survive beyond 24 h due to acid crash (see the text).

�K of Clostridium acetobutylicum Plays Dual Roles

January 2014 Volume 196 Number 2 jb.asm.org 291

on April 29, 2016 by guest

http://jb.asm.org/

Dow

nloaded from

�sigK, WT, and SigK_comp strains was investigated to examinehow these factors might be affected by sigK deletion and comple-mentation. It is worth noting that for the �sigK strain, double theamount of protein was loaded for the Spo0A Western blot than forthe WT strain. This was to ensure sufficient amounts of proteins todetect small, if any, amounts of Spo0A. No bands were detectedfor either Spo0A or �G, while a weak band was detected for �F (Fig.3A). The latter is consistent with prior findings that sigF is tran-scribed from a �H-dependent promoter in both B. subtilis (42) andC. difficile (P. difficile) (43). Spo0A acts as a strong enhancer of sigFtranscription in B. subtilis (44). This model is also supported bytranscriptional studies with C. acetobutylicum that revealed thatsigF was downregulated in SKO1 (the spo0A inactivation mutant[15]) �8-fold relative to the WT (45) and the finding that inSKO1, a weak �F protein band is also detected (see Fig. S2 in thesupplemental material). Finally, in this and other sequenced Clos-tridium genomes, a �H-binding motif can be identified in the pro-moter region of the sigF operon (46). Consistent with the pheno-type observed in the �sigK mutant (i.e., sporulation blocked priorto asymmetric division and the production of very low levels ofsolvents), these data suggest that sigK deletion effectively abolishesSpo0A and �G production while markedly reducing �F produc-tion. Due to the loss of activity of our antibody against �E, we wereunable to examine its protein level. Thus, sqRT-PCR was carriedout for sigE expression in the �sigK strain after 26 h of culture(when sigE expression is typically the highest [9]), and no tran-script could be detected (Fig. 3C).

Protein production of Spo0A, �F, and �G was restored in the

SigK_comp strain (Fig. 3A). Since, as mentioned earlier, theSigK_comp strain was able to successfully sporulate, it is safe toassume that �E production was also restored in the complemen-tation strain; otherwise it would not have been able to form viablespores (6). To provide further confirmation and more insights asto the role of �K in early sporulation, we used qRT-PCR analysis toprobe the mRNA levels of these and other related genes.

Deletion of sigK significantly reduces mRNA levels of spo0A,sigF, sigE, sigG, spoIIE, and adhE1 (aad), but sigH expressionremains largely unaffected. Based on the data from the Westernanalysis and TEM imaging, and since the �sigK mutant producedonly small amounts of solvents, we expected that transcript levelsof the major sporulation-related genes as well as of adhE1 wouldbe downregulated in the �sigK mutant. adhE1, or aad (ca_p0162),which is the canonical solventogenic gene of this organism andwhich codes for a bifunctional acetaldehyde/alcohol dehydroge-nase (AAD), is the first gene of the tricistronic sol operon (adhE1-ctfAB), which is known to be largely controlled by Spo0A (15,47–50). While the data presented above clearly show that �K isnecessary for Spo0A production, an additional key question wewanted to address is if �K affects the expression of the gene for thefirst sporulation-specific sigma factor, sigH. Thus, we used tem-poral qRT-PCR analysis (four time points— 6 h, 12 h, 20 h, and 26h—to probe the expression of sporulation factors involved instages 0, I, II, and III) to examine the impact of sigK silencing onthe mRNA levels of the major sporulation-related genes (sigH,spo0A, spoIIE, sigF, sigE, and sigG) and of adhE1 (Fig. 3D). Con-sistent with the Western blots, the data revealed that spo0A was

FIG 3 Protein and transcript levels of sporulation and solvent formation genes are severely downregulated in the �sigK mutant. Shown are Western blots frombiological replicates (a and b) for Spo0A, �F, and �G in the �sigK, WT, and SigK_comp (A) and �sigK p94Spo0A (B) strains at 32 h and 45 h. All Western blotswere loaded with 30 �g of total protein, except that for the Spo0A WT and �sigK p94Spo0A strains, the blots were loaded with 15 �g and 5 �g, respectively. (C)The sigE transcript is detected in the �sigK p94Spo0A at 24 h from biological replicates (a and b), while no sigE transcript was detected after 26 h for the �sigKstrain from biological replicates (a and b). �, reactions in which reverse transcriptase was added; �, reactions in which no reverse transcriptase was added. M,2-log ladder (NEB). (D) qRT-PCR data showing the relative fold change of the major sporulation-related genes as well as adhE1 in the �sigK mutant relative tothe WT strain at 6 h, 12 h, 20 h, and 26 h. All experiments were done in two biological replicates. Error bars represent SEMs. Statistical significance was tested witha two-sample t test (*, P 0.05).

Al-Hinai et al.

292 jb.asm.org Journal of Bacteriology

on April 29, 2016 by guest

http://jb.asm.org/

Dow

nloaded from

downregulated (�sigK versus WT) �9-, �51-, and � 46-fold at 12h, 20 h, and 26 h, respectively. sigF was downregulated �13-,�30-, and �20-fold at 12, 20, and 26 h, respectively, which isconsistent with the Western blot data (Fig. 3D) and the priorfinding that Spo0A plays an important role in the expression ofsigF (9, 45, 51). Expression of sigE showed a strong downregula-tion, �53-fold and �131-fold, at 20 h and 26 h, respectively (Fig.3D). In contrast, expression of sigH (ca_c3152), the first sporula-tion-specific sigma factor upstream of spo0A expression, was notsignificantly impacted by sigK silencing during the transitionalphase (12 h), which is the period that matters for this investiga-tion. Expression of sigH typically commences at or prior to 6 h andprecedes Spo0A expression and activation in this organism (9).The modest (4- to 7-fold) sigH downregulation at the later timepoints likely reflects secondary impacts. All other sporulationgenes examined (spoIIE and sigG) as well adhE1 were consistentlyand severely downregulated at all time points. For example, adhE1was downregulated �20-fold at 12 h. This is consistent with datafrom the SKO1 strain, the spo0A inactivation strain (15), whichalso displays severe adhE1 downregulation.

Taken together, these data show that �K is necessary for Spo0Aproduction and activity, and thus, sigK disruption abolishes spo-rulation by blocking it at the spo0A expression stage, without ap-parently affecting sigH expression during the transitional phase.That sigK is expressed early, prior to spo0A expression, is an un-expected finding contrary to the B. subtilis model and not antici-pated even by the recent study with C. perfringens (12). In the C.botulinum sigK disruption mutant, the spo0A transcript wasdownregulated �5-fold relative to the WT, but it is unclear if theSpo0A protein was completely absent, since no Western blots werepresented (13). To confirm early sigK expression, we examined itsmRNA profile as described below.

sigK is expressed very early in exponential phase at low levelsand is strongly upregulated during the middle and late station-ary phases. Using sqRT-PCR analysis, we confirmed that sigK isexpressed early in exponential phase (6 h; A600 of 0.5), prior to anysignificant production or activation of Spo0A (9). RNA was ex-tracted and cDNA was generated from both the WT and �sigK(negative control) strains, and sqRT-PCR was carried out withsigK-specific primers (see Table S1 in the supplemental material).A band of the expected size (641 bp) was visible for the WT strain,while, as expected, none appeared for the �sigK strain (Fig. 4A).These data indicate that the sigK transcript is indeed present earlyduring the life cycle of C. acetobutylicum.

Additionally, we wanted to investigate the presence of the sigKtranscript as early as we possibly could following heat shocking ofWT cultures and germination. Thus, we heat shocked 10 samplesof WT spores to germinate them in 10 ml of CGM, allowed themto reach an optical density at 600 nm (OD600) of �0.03 (repre-senting an extremely early exponential growth phase of primarygerminated culture), and combined them to have enough cells forextracting sufficient amounts of RNA for sqRT-PCR analysis.Subsequently, sqRT-PCR was carried out, and the sigK transcriptwas clearly detected (Fig. 4B). These data show that sigK is tran-scribed very early following the germination of the spores. Next,we examined the temporal expression profile of sigK in WT C.acetobutylicum by qRT-PCR analysis in two biological replicates(Fig. 4C). RNA extraction and cDNA generation were preparedfrom duplicate WT cultures at 6 h, 15 h, 24 h, 30 h, 36 h, and 48 h.The 24-h time point showed the lowest expression levels and was

thus used as a reference point for qRT-PCR analysis. The resultsshow that sigK is expressed at low levels during exponential phaseand at very high levels in the middle and late stationary phases.The data also suggest that it may follow a biphasic pattern (Fig.4C). However, our data suggest that during the exponential (6 h)and transition (15 h) phases, sigK expression is probably higherthan that at the 24-h time point (early stationary phase) (Fig. 4C).After a drop in expression at 24 h, sigK expression gradually in-creased. At 30 h, 36 h, and 48 h (time points in the middle and latestationary phases), sigK expression increased �1.2-fold, �3.4-fold, and �12-fold, respectively, relative to the 24-h expression

FIG 4 Very early and two-phase expression of sigK in WT C. acetobutylicumand downregulation of cotS and yabG in the �sigK mutant. (A) sqRT-PCRshowing early expression of sigK at 6 h (A600, �0.5). Data from two biologicalreplicate experiments (a and b) are shown for both the WT and �sigK strains.M, 2-log ladder (NEB). (B) Very early expression of sigK following heat shock-ing of WT colonies and germination at an OD of �0.03. Ten WT cultures wereheat shocked and combined into one tube to allow for enough material toextract RNA. �, reactions in which reverse transcriptase was added; �, reac-tions in which no reverse transcriptase was added. M, 2-log ladder (NEB). (C)qRT-PCR showing the biphasic expression pattern of sigK, low expressionearly followed by the gradual increase of expression after 24 h. All experimentswere done in two biological replicates. Error bars represent SEM. Statisticalsignificance was tested with a two-sample t test (*, P 0.05; **, P 0.10). (D)Fold expression of cotS and yabG in �sigK. cotS and yabG are both downregu-lated in the �sigK strain relative to the WT up to �3.5-fold and �7-fold,respectively. Error bars represent SEMs. Statistical significance was tested witha two-sample t test (*, P 0.05; **, P 0.10).

�K of Clostridium acetobutylicum Plays Dual Roles

January 2014 Volume 196 Number 2 jb.asm.org 293

on April 29, 2016 by guest

http://jb.asm.org/

Dow

nloaded from

level for replicate A (Fig. 4C). For replicate B, the same patterncould be observed and sigK expression increased �2-fold, �8-fold, and �56-fold at 30 h, 36 h, and 48 h, respectively (Fig. 4C).These differences were statistically significant.

Our data so far have established that �K is necessary for sporu-lation by controlling Spo0A production. Is the late upregulation ofsigK (Fig. 4C) an inconsequential anomaly, or does it play a secondrole in late sporulation? This has not been entertained or probedin either C. perfringens (12) or C. botulinum (13), and if provenright, �K will be the first known sporulation-specific sigma factorwith two, developmentally separated, roles in sporulation. This isexamined next.

�K is necessary for terminal-stage sporulation: expression ofspo0A from the ptb promoter restores solventogenesis andSpo0A, �F, �E, and �G production but not sporulation in the�sigK strain. The difficulty in establishing a role for �K late insporulation is that sigK deletion abolishes Spo0A production (Fig.3A), and thus, all later stages of sporulation are also abolished. Wereasoned that if we expressed spo0A in the �sigK strain from adifferent, early expression promoter, we might succeed in restor-ing Spo0A production, activity, and early sporulation. If sporula-tion were completely restored, then �K would not be necessary forthe later stages of sporulation. If, on the other hand, sporulationwere blocked later in sporulation, presumably past the �G-con-trolled stage (6), then this would show that the dramatic, late sigKupregulation (Fig. 4C) is of physiological importance. For earlyspo0A expression without its natural promoter, we used the ptbpromoter (52) of the ptb-buk operon, which enables formation ofbutyrate, a metabolite necessary for the growth of this organism.Solvent formation is enabled by the expression of the sol operon(50, 53), which is controlled by the activated (phosphorylated)Spo0A (9, 15, 54). Thus, we used solvent formation as a first assayto assess restoration of Spo0A activity when we expressed spo0Aon plasmid p94Spo0A from the ptb promoter. Our data show that�sigK p94Spo0A cultures are indeed able to produce normal levelsof solvents, with final titers of 144 mM, 85 mM, and 21 mMbutanol, acetone, and ethanol, respectively (Table 2). The acetateand butyrate levels were reduced to 1 mM and 12 mM, respec-tively, which are similar to those in the WT without pH control

(Table 2). These data suggest that Spo0A was activated and that �K

is not necessary for Spo0A activation, although it is necessary, asshown above (Fig. 3A and D), for Spo0A mRNA and protein pro-duction. We also examined the effects of spo0A overexpression incells of the WT p94Spo0A strain, and the data indicated that thisstrain produced solvent levels that were comparable to those in theWT strain (Table 2).

Yet the �sigK p94Spo0A mutant was unable to form anyspores, as evidenced by the data obtained from the spore assays(Fig. 2A) as well as spore staining (see Fig. S1 in the supplementalmaterial). The WT p94Spo0A strain produced normal levels ofspores and vegetative cells, and thus, it appears the spo0A overex-pression does not have any adverse effects on sporulation in WTcultures (Fig. 2A). To probe the stage where sporulation wasblocked in the �sigK p94Spo0A strain, we examined the strain’smorphological characteristics by TEM analysis (Fig. 5). TEM im-ages showed that this strain was able to initiate asymmetric divi-sion and produce granulose, but the forespore structure andmembrane were ill formed. A spore coat is either absent or veryundeveloped, while the spore membrane appears crimped andruptured (Fig. 5). Still, these TEM images suggest a more ad-vanced developmental stage for the �sigK p94Spo0A strain thanfor the sigG inactivation strain (6). These data suggest, then, thatSpo0A, �F, �E, and �G expression was restored in the �sigKp94Spo0A strain. Granulose formation is controlled by �E in thisorganism (6); thus, �E production must have been restored in thisstrain, and this was confirmed by sqRT-PCR analysis (Fig. 3C).Western analysis (Fig. 3B) confirmed the production of Spo0A,�F, and �G to levels similar to those in the WT strain.

Taken together, these data show that in addition to enablingSpo0A production, which is necessary for initiating the sporula-tion cascade, �K is also necessary for the late stages of sporulation,past the �G stage, and is apparently necessary for spore coat for-mation. We thus used qRT-PCR analysis to examine the expres-sion of cotS and ca_c2905 (yabG), which are involved in spore coatassembly and are controlled by �K in B. subtilis (55, 56). The re-sults showed that at 20 h and 26 h, the cotS mRNA was downregu-lated �2- and �4-fold, respectively, in the �sigK strain relative tothe WT (Fig. 4D). Similarly, the mRNA levels of ca_c2905 (yabG)

FIG 5 TEM images of the �sigK p94Spo0A strain after 5 days of culture. The cells appear to be releasing an immature and underdeveloped spore (I). The sporemembrane appeared to be crimped and ill formed (II). The strain was able to produce granulose vesicles (III) but showed what appears to be a rupturedmembrane (IV) with no identifiable spore coat.

Al-Hinai et al.

294 jb.asm.org Journal of Bacteriology

on April 29, 2016 by guest

http://jb.asm.org/

Dow

nloaded from

were downregulated �4- and �7-fold at 20 h and 26 h, respec-tively, in the �sigK mutant (Fig. 4D). Additionally, bioinformaticsanalysis predicted a strong �K-like consensus binding sequence(18) upstream of their respective start codons (see Table S2 in thesupplemental material).

Next, we examined which sigma factors might control the latedramatic upregulation of sigK (Fig. 4C) and its posttranslationalprocessing.

sigK expression is �E dependent, while the expression ofspoIVB and spoIVFB is �G dependent. We have thus far demon-strated the involvement of �K in both early and late stages of spo-rulation in C. acetobutylicum. Next we wanted to examine thefactors that might control sigK expression and its posttranslationalactivation during the later stages of sporulation. Previous workhas identified the transcription start site (TSS) of sigK in C. aceto-butylicum, and both �E and �K--like binding sequences were pre-dicted in its promoter region (57). Thus, we hypothesized thatexpression of sigK is, partly at least, dependent on �E before �K

becomes active and regulates its own expression. To examine thetranscriptional profile of sigK mRNA in EKO1 (sigE mutant) ver-sus the WT, qRT-PCR was carried out. The data showed that thesigK mRNA was downregulated �84-fold at 12 h (transitionalphase) in the EKO1 strain (Fig. 6A), strongly suggesting that �E isneeded for the initial transcription of sigK. In the B. subtilis model,since sigK is initially translated in the inactive form, pro-�K, itcannot regulate its own expression before the prosequence iscleaved by the action of the SpoIVFB protease via a SpoIVB-dependent pathway (11). Global transcriptional analysis of theGKO1 strain (sigG mutant) showed that sigK, spoIVB, andspoIVFB were all downregulated, up to �6-fold, �3-fold, and�4-fold, respectively, in the GKO1 strain relative to the WT (Fig.6B). Additionally, time course gene expression analysis by qRT-PCR of spoIVB and spoIVFB in duplicate WT cultures showed adramatic upregulation during late stationary phase relative to thefirst time point (6 h) (Fig. 6C). Since �E is expressed in the GKO1strain, these data suggest that the expression of spoIVFB and thatof spoIVB are both �G dependent. In contrast, only spoIVB expres-sion was shown to be regulated by �G in B. subtilis, while spoIVFBis �E dependent (10). Thus, the downregulation of sigK in theGKO1 strain maybe be a secondary effect due to the inefficientprocessing of pro-�K to the mature �K and hence its inability toregulate its own expression. This of course assumes that the B.subtilis model is valid in C. acetobutylicum. Taken together, thesedata suggest that the late expression of sigK is �E dependent before�G activates the expression of spoIVB and spoIVFB, whose prod-ucts may subsequently process the pro-�K to the mature �K, thusallowing it to regulate its own expression as it does in B. subtilis(10).

DISCUSSION

sigK was successfully deleted in C. acetobutylicum by precise allelicexchange via homologous recombination, thereby generating the�sigK and �sigK_um (unmarked) strains. The �sigK strain wasrescued by successfully integrating sigK back in its naturalgenomic locus. Plasmid-based complementation of sigK was notsuccessful in rescuing �sigK, thus suggesting that high sigK mRNAlevels are counterproductive. The data presented here establish anovel and unique role for �K in the sporulation cascade of C.acetobutylicum: a role with two developmentally separated parts,one role upstream of Spo0A during early sporulation and one

downstream of �G during late sporulation. There is no precedentfor such a role for any sporulation-specific sigma factors amongendospore formers. Like in C. perfringens and C. botulinum, �K

appears to be involved in early stages of sporulation in C. acetobu-tylicum, which is in stark contrast to its exclusive role during thelate stages of sporulation in B. subtilis (12, 13, 27) or its recently

FIG 6 Transcriptional profiling of sigK, spoIVFB, and spoIVB in either EKO1or GKO1. (A) Expression of sigK in EKO1 is downregulated 84-fold relative tothe WT during the transitional phase and �4-fold during the stationary phase.(B) Microarray data showing expression fold difference of spoIVFB, spoIVB,and sigK in GKO1 relative to the WT. The fold difference shown is the averageof two time points (28 h and 30 h), which correspond to maximum sigGexpression in the WT (9). (C) Time course gene expression profiling ofspoIVFB and spoIVB in WT cells relative to the first time point. All experimentswere done in two biological replicates. The data show a dramatic upregulationof both genes during the late stages of sporulation coinciding with the in-creased expression of sigK, thus indicating that they may possibly play a role inits processing at the posttranslational level. Error bars represent SEMs. Statis-tical significance was tested with a two-sample t test (*, P 0.05).

�K of Clostridium acetobutylicum Plays Dual Roles

January 2014 Volume 196 Number 2 jb.asm.org 295

on April 29, 2016 by guest

http://jb.asm.org/

Dow

nloaded from

proposed role in C. difficile (P. difficile) (28). In order to demon-strate the role of �K in late sporulation, we had to bypass the initialinvolvement of �K in the sporulation cascade. We did so by ex-pressing spo0A from the strong and early ptb promoter in the�sigK strain, thus demonstrating that �K plays a critical role in latesporulation, as supported by the nonsporulating phenotype of the�sigK p94Spo0A strain.

Comparison of TEM images (see Fig. S3 in the supplementalmaterial) between the �sigK, FKO1 (sigF inactivation strain) (8),EKO1 (sigE inactivation strain) (6), and GKO1 (sigG inactivationstrain) (6) mutants reveals that the �sigK strain has its sporulationblocked at the same stage (i.e., prior to stage II) as FKO1 but at anearlier stage than either the EKO1 or GKO1 strain. Thus, thesedata support the conclusion that �K plays an essential role duringthe early stages of sporulation. On the other hand, the �sigKp94Spo0A strain appeared to progress further in the sporulationprocess than the GKO1 strain but was still unable to form viablespores (Fig. 5), thus showing that �K is also necessary during thelate stages of sporulation.

It was suggested that in C. perfringens, early expression of sigKduring the exponential and stationary phases is driven from anunknown promoter either within or upstream of the gene(CPR_1739; coding for a putative penicillin binding protein) im-mediately upstream of sigK (12). In C. acetobutylicum, sigK is lo-cated immediately downstream of ca_c1688 (also coding for a pu-tative penicillin binding protein), and thus, it is possible that itsearly expression is controlled in the same manner as in C. perfrin-gens. However, this genomic organization of the sigK locus is notconserved in C. botulinum, as sigK is located 425 bp downstreamof the adjacent gene, which encodes a small hypothetical protein(58). This would suggest that there is another mechanism for theearly expression of sigK in C. botulinum and possibly other clos-tridia. An alternative mechanism would be that early expression ofsigK is driven from a �A promoter, since a �A-consensus binding

sequence can be identified in the sigK promoter of C. acetobutyli-cum (see Fig. S4 in the supplemental material). Regardless, animportant question remains: how is �K processed and activatedearly? If one assumes that the B. subtilis model applies here as well,are SpoIVB and SpoIVFB produced early, albeit at very low levels,thus processing pro-�K to the mature �K, or could there be an-other explanation? One possibility is that �K is passed down to thespore and subsequently the vegetative cell via epigenetic inheri-tance (59). In fact, a study with B. subtilis shows that the signal toinitiate the sporulation process (e.g., the sporulation phosphore-lay system) is present during exponential growth and appears tobe epigenetically passed on to the next generation (60). However,at this time one cannot rule out the possibility that pro-�K mayhave different promoter specificity than the mature �K, whichcould also help explain the early versus the late roles of �K. More-over, it was shown that in B. subtilis, these sporulation-specificsigma factors are not always confined to the compartment inwhich they are predominantly needed in the developing endo-spore or mother cell. For example, it was shown that �G becomesactive in the mother cell (61), although it is a prespore-specificsigma factor. It is worth noting that these experiments were donewith certain sigG mutants of B. subtilis that had abnormal sigGexpression. Nonetheless, the experiments demonstrated that �G

could be active in the mother cell rather than the prespore. Couldit be that �K behaves in a similar manner by becoming active in theprespore and subsequently inherited in its active form in the ma-ture spore? Then, once the spore starts to germinate, �K may“jump start” the expression of spo0A, either directly or indirectly.Indeed, a �K-like consensus binding sequence was identified,albeit with a slightly bigger spacer sequence, in the spo0A pro-moter (see Fig. S5 in the supplemental material).

We thus propose a modified sporulation cascade model for C.acetobutylicum (Fig. 7) which, based on data reported so far in theliterature and discussed above, could also be valid for other Clos-

FIG 7 Proposed sporulation cascade model in C. acetobutylicum. Gray dashed lines indicate a hypothesized interaction between �E and �G. The figure wasconstructed from data reported in references 6, 8, and 46.

Al-Hinai et al.

296 jb.asm.org Journal of Bacteriology

on April 29, 2016 by guest

http://jb.asm.org/

Dow

nloaded from

tridium organisms, although not C. difficile (P. difficile) (28). Ac-cording to this model, the positive-feedback loop, between Spo0Aand �K, ensures that the signal to sporulate remains strong duringthe sporulation cycle. Sporulation in B. subtilis requires a highthreshold level of Spo0A production (44), and this is likely valid inC. acetobutylicum based on detailed transcriptional data (9),whereby all major sigma factors display a bimodal pattern of ex-pression, low early expression followed by strong upregulationlater. Thus, the proposed model provides a mechanism by whichthe required threshold expression level of the sporulation-relatedgenes is maintained at high enough levels to ensure successfulcompletion of sporulation in C. acetobutylicum. Yet it is wellknown that sporulation does not always take place at a high fre-quency in this (62) and other solventogenic Clostridium organ-isms, and this would suggest a weak link in the feedback loop ofFig. 7. Of note here is the so-called strain degeneration issue,which has been known since Pasteur’s time. Namely, continuous,vegetative transfers of solventogenic Clostridium organisms leadto greatly diminished abilities to produce solvents and to sporu-late (53). Degeneration can be permanent, as in the case of C.acetobutylicum, whereby the genes for solvent formation are car-ried on the pSOL1 megaplasmid, which when lost leads to a per-manent asporogenous strain that produces no solvents (53). Inmost other solvetogenic Clostridium organisms, however, the sol-ventogenic genes are carried on the chromosome; thus, a differentmechanism must account for the degeneration process, which inmost cases can be, partially at least, restored by altering cultureconditions (53). Significantly, starting cultures by heat shockingspores almost invariably guarantees good solvent production andreasonable sporulation (53). One possibility is that this degenera-tion phenotype derives from the bistability of the system due tothe positive-feedback circuit (Fig. 7) that is generated by thebiphasic action of �K. Bistability requires an appropriate feedbackcircuit (like a positive-feedback one), a nonlinearity which is partof this circuit, and a “good” balance between the two legs of thefeedback loop (63). Bistability has been well established for B.subtilis, whereby the bistable phenotype (“sporulate” versus “donot sporulate”) derives from the positive feedback of phosphory-lated Spo0A on its own transcription and the nonlinear dynamicsintroduced by the phosphorelay system (Spo0F and Spo0B),which is part of this positive-feedback circuit (59). There is nophosphorelay system in Clostridium organisms (11), but the pos-itive-feedback circuit of Fig. 7 contains many more nonlinearitiesdue to gene expression of sigma and other sporulation factors andthe associated protein activation events.

ACKNOWLEDGMENTS

This work was supported in part by U.S. Department of Energy Electro-fuels ARPA-E program DE-FOA-0000206 under Award DE-AR0000059.Financial support for Mohab Al-Hinai was provided by the government ofOman.

We thank Bryan Tracy for insightful discussions and suggestions. Wealso thank Mason Smith and Alexander Jones for assistance with bioreac-tor experiments and cDNA generation. We acknowledge assistance byShannon Modla and the use of the Delaware Biotechnology Institute Bio-Imaging Facility for TEM analysis.

REFERENCES1. Nolling J, Breton G, Omelchenko MV, Makarova KS, Zeng QD, Gibson R,

Lee HM, Dubois J, Qiu DY, Hitti J, GTC Sequencing Center Production,Finishing, and Bioinformatics Teams, Wolf YI, Tatusov RL, Sabathe F,

Doucette-Stamm L, Soucaille P, Daly MJ, Bennett GN, Koonin EV, SmithDR. 2001. Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum. J. Bacteriol. 183:4823–4838. http://dx.doi.org/10.1128/JB.183.16.4823-4838.2001.

2. Papoutsakis ET. 2008. Engineering solventogenic clostridia. Curr. Opin.Biotechnol. 19:420 – 429. http://dx.doi.org/10.1016/j.copbio.2008.08.003.

3. Lee SY, Park JH, Jang SH, Nielsen LK, Kim J, Jung KS. 2008. Fermen-tative butanol production by clostridia. Biotechnol. Bioeng. 101:209 –228.http://dx.doi.org/10.1002/bit.22003.

4. Tracy BP, Jones SW, Fast AG, Indurthi DC, Papoutsakis ET. 2012.Clostridia: the importance of their exceptional substrate and metabolitediversity for biofuel and biorefinery applications. Curr. Opin. Biotechnol.23:364 –381. http://dx.doi.org/10.1016/j.copbio.2011.10.008.

5. Steiner E, Dago AE, Young DI, Heap JT, Minton NP, Hoch JA, YoungM. 2011. Multiple orphan histidine kinases interact directly with Spo0A tocontrol the initiation of endospore formation in Clostridium acetobuty-licum. Mol. Microbiol. 80:641– 654. http://dx.doi.org/10.1111/j.1365-2958.2011.07608.x.

6. Tracy BP, Jones SW, Papoutsakis ET. 2011. Inactivation of sigma(E) andsigma(G) in Clostridium acetobutylicum illuminates their roles in clos-tridial-cell-form biogenesis, granulose synthesis, solventogenesis, andspore morphogenesis. J. Bacteriol. 193:1414 –1426. http://dx.doi.org/10.1128/JB.01380-10.

7. Bi CH, Jones SW, Hess DR, Tracy BP, Papoutsakis ET. 2011. SpoIIE isnecessary for asymmetric division, sporulation, and expression of sig-ma(F), sigma(E), and sigma(G) but does not control solvent productionin Clostridium acetobutylicum ATCC 824. J. Bacteriol. 193:5130 –5137.http://dx.doi.org/10.1128/JB.05474-11.

8. Jones SW, Tracy BP, Gaida SM, Papoutsakis ET. 2011. Inactivation ofsigma(F) in Clostridium acetobutylicum ATCC 824 blocks sporulationprior to asymmetric division and abolishes sigma(E) and sigma(G) pro-tein expression but does not block solvent formation. J. Bacteriol. 193:2429 –2440. http://dx.doi.org/10.1128/JB.00088-11.

9. Jones SW, Paredes CJ, Tracy B, Cheng N, Sillers R, Senger RS, Papout-sakis ET. 2008. The transcriptional program underlying the physiology ofclostridial sporulation. Genome Biol. 9:R114. http://dx.doi.org/10.1186/gb-2008-9-7-r114.

10. Piggot PJ, Hilbert DW. 2004. Sporulation of Bacillus subtilis. Curr. Opin.Microbiol. 7:579 –586. http://dx.doi.org/10.1016/j.mib.2004.10.001.

11. Paredes CJ, Alsaker KV, Papoutsakis ET. 2005. A comparative genomicview of clostridial sporulation and physiology. Nat. Rev. Microbiol.3:969 –978. http://dx.doi.org/10.1038/nrmicro1288.

12. Harry KH, Zhou R, Kroos L, Melville SB. 2009. Sporulation and entero-toxin (CPE) synthesis are controlled by the sporulation-specific sigmafactors SigE and SigK in Clostridium perfringens. J. Bacteriol. 191:2728 –2742. http://dx.doi.org/10.1128/JB.01839-08.

13. Kirk DG, Dahlsten E, Zhang Z, Korkeala H, Lindstrom M. 2012.Involvement of Clostridium botulinum ATCC 3502 sigma factor K inearly-stage sporulation. Appl. Environ. Microbiol. 78:4590 – 4596. http://dx.doi.org/10.1128/AEM.00304-12.

14. Dürre P, Bohringer M, Nakotte S, Schaffer S, Thormann K, Zickner B.2002. Transcriptional regulation of solventogenesis in Clostridium aceto-butylicum. J. Mol. Microbiol. Biotechnol. 4:295–300.

15. Harris LM, Welker NE, Papoutsakis ET. 2002. Northern, morphological,and fermentation analysis of spo0A inactivation and overexpression inClostridium acetobutylicum ATCC 824. J. Bacteriol. 184:3586 –3597.http://dx.doi.org/10.1128/JB.184.13.3586-3597.2002.

16. Ravagnani A, Jennert KCB, Steiner E, Grunberg R, Jefferies JR, Wilkin-son SR, Young DI, Tidswell EC, Brown DP, Youngman P, Morris JG,Young M. 2000. Spo0A directly controls the switch from acid to solventproduction in solvent-forming clostridia. Mol. Microbiol. 37:1172–1185.http://dx.doi.org/10.1046/j.1365-2958.2000.02071.x.

17. Siranosian KJ, Grossman AD. 1994. Activation of spo0A transcription bysigma H is necessary for sporulation but not for competence in Bacillussubtilis. J. Bacteriol. 176:3812–3815.

18. Haldenwang WG. 1995. The sigma factors of Bacillus subtilis. Microbiol.Rev. 59:1–30.

19. de Hoon MJL, Eichenberger P, Vitkup D. 2010. Hierarchical evolutionof the bacterial sporulation network. Curr. Biol. 20:R735–R745. http://dx.doi.org/10.1016/j.cub.2010.06.031.

20. Feucht A, Evans L, Errington J. 2003. Identification of sporulation genesby genome-wide analysis of the sigma(E) regulon of Bacillus subtilis. Mi-crobiology 149:3023–3034. http://dx.doi.org/10.1099/mic.0.26413-0.

�K of Clostridium acetobutylicum Plays Dual Roles

January 2014 Volume 196 Number 2 jb.asm.org 297

on April 29, 2016 by guest

http://jb.asm.org/

Dow

nloaded from

21. Li ZS, Di Donato F, Piggot PJ. 2004. Compartmentalization of geneexpression during sporulation of Bacillus subtilis is compromised in mu-tants blocked at stage III of sporulation. J. Bacteriol. 186:2221–2223. http://dx.doi.org/10.1128/JB.186.7.2221-2223.2003.

22. Illing N, Errington J. 1991. Genetic regulation of morphogenesis inBacillus subtilis—roles of sigma E and sigma F in prespore engulfment.J. Bacteriol. 173:3159 –3169.

23. Kroos L, Kunkel B, Losick R. 1989. Switch protein alters specificity ofRNA polymerase containing a compartment-specific sigma factor. Sci-ence 243:526 –529. http://dx.doi.org/10.1126/science.2492118.

24. Cutting S, Oke V, Driks A, Losick R, Lu S, Kroos L. 1990. A foresporecheckpoint for mother cell gene expression during development in B. subtilis.Cell 62:239–250. http://dx.doi.org/10.1016/0092-8674(90)90362-I.

25. Karmazyn-Campelli C, Bonamy C, Savelli B, Stragier P. 1989. Tandemgenes encoding sigma-factors for consecutive steps of development inBacillus subtilis. Genes Dev. 3:150 –157. http://dx.doi.org/10.1101/gad.3.2.150.

26. Li J, McClane BA. 2010. Evaluating the involvement of alternative sigmafactors SigF and SigG in Clostridium perfringens sporulation and entero-toxin synthesis. Infect. Immun. 78:4286 – 4293. http://dx.doi.org/10.1128/IAI.00528-10.

27. Farquhar R, Yudkin MD. 1988. Phenotypic and genetic characterizationof mutations in the spoIVC locus of Bacillus subtilis. J. Gen. Microbiol.134:9 –17.

28. Fimlaid KA, Bond JP, Schutz KC, Putnam EE, Leung JM, Lawley TD,Shen A. 2013. Global analysis of the sporulation pathway of Clostridiumdifficile. PLoS Genet. 9:e1003660. http://dx.doi.org/10.1371/journal.pgen.1003660.

29. Yutin N, Galperin MY. 2013. A genomic update on clostridial phylogeny:Gram-negative spore formers and other misplaced clostridia. Environ.Microbiol. 15:2631–2641. http://dx.doi.org/10.1111/1462-2920.12173.

30. Al-Hinai MA, Fast AG, Papoutsakis ET. 2012. Novel system for efficientisolation of Clostridium double-crossover allelic exchange mutants en-abling markerless chromosomal gene deletions and DNA integration.Appl. Environ. Microbiol. 78:8112– 8121. http://dx.doi.org/10.1128/AEM.02214-12.

31. Desai RP, Papoutsakis ET. 1999. Antisense RNA strategies for metabolicengineering of Clostridium acetobutylicum. Appl. Environ. Microbiol.65:936 –945.

32. Mermelstein LD, Papoutsakis ET. 1993. In vivo methylation in Esche-richia coli by the Bacillus subtilis phage phi 3T I methyltransferase toprotect plasmids from restriction upon transformation of Clostridiumacetobutylicum ATCC 824. Appl. Environ. Microbiol. 59:1077–1081.

33. Mermelstein LD, Welker NE, Bennett GN, Papoutsakis ET. 1992. Ex-pression of cloned homologous fermentative genes in Clostridium aceto-butylicum ATCC 824. Biotechnology (NY) 10:190 –195. http://dx.doi.org/10.1038/nbt0292-190.

34. Sillers R, Al-Hinai MA, Papoutsakis ET. 2009. Aldehyde-alcohol dehy-drogenase and/or thiolase overexpression coupled with CoA transferasedownregulation lead to higher alcohol titers and selectivity in Clostridiumacetobutylicum fermentations. Biotechnol. Bioeng. 102:38 – 49. http://dx.doi.org/10.1002/bit.22058.

35. Alsaker KV, Paredes CJ, Papoutsakis ET. 2005. Design, optimization andvalidation of genomic DNA microarrays for examining the Clostridiumacetobutylicum transcriptome. Biotechnol. Bioprocess Eng. 10:432– 443.http://dx.doi.org/10.1007/BF02989826.

36. Schaeffer AB, Fulton MD. 1933. A simplified method of staining en-dospores. Science 77:194. http://dx.doi.org/10.1126/science.77.1990.194.

37. Tomas CA, Welker NE, Papoutsakis ET. 2003. Overexpression of groESLin Clostridium acetobutylicum results in increased solvent production andtolerance, prolonged metabolism, and changes in the cell’s transcriptionalprogram. Appl. Environ. Microbiol. 69:4951– 4965. http://dx.doi.org/10.1128/AEM.69.8.4951-4965.2003.

38. Paredes CJ, Senger RS, Spath IS, Borden JR, Sillers R, Papoutsakis ET.2007. A general framework for designing and validating oligomer-basedDNA microarrays and its application to Clostridium acetobutylicum.Appl. Environ. Microbiol. 73:4631– 4638. http://dx.doi.org/10.1128/AEM.00144-07.

39. Smyth GK. 2005. Limma: linear models for microarray data, p 397– 420.In Gentleman R, Carey V, Dudoit S, Irizarry RA, Huber W (ed), Bioinfor-matics and computational biology solutions using R and Bioconductor.Springer, New York, NY.

40. Yang YH, Dudoit S, Luu P, Speed TP. 2001. Normalization for cDNA

microarray data, p 141–152. In Bittner ML, Chen Y, Dorsel AN, Dough-erty ER (ed), Proceedings of SPIE, vol 4266. Microarrays: optical technol-ogies and informatics. SPIE, Bellingham, WA.

41. Maddox IS, Steiner E, Hirsch S, Wessner S, Gutierrez NA, Gapes JR,Schuster KC. 2000. The cause of “acid-crash” and “acidogenic fermenta-tions” during the batch acetone-butanol-ethanol (ABE-) fermentationprocess. J. Mol. Microbiol. Biotechnol. 2:95–100.

42. Wu JJ, Howard MG, Piggot PJ. 1989. Regulation of transcription of theBacillus subtilis spoIIA locus. J. Bacteriol. 171:692– 698.

43. Saujet L, Monot M, Dupuy B, Soutourina O, Martin-Verstraete I. 2011.The key sigma factor of transition phase, SigH, controls sporulation, me-tabolism, and virulence factor expression in Clostridium difficile. J. Bac-teriol. 193:3186 –3196. http://dx.doi.org/10.1128/JB.00272-11.

44. Fujita M, Gonzalez-Pastor JE, Losick R. 2005. High- and low-thresholdgenes in the Spo0A regulon of Bacillus subtilis. J. Bacteriol. 187:1357–1368. http://dx.doi.org/10.1128/JB.187.4.1357-1368.2005.

45. Tomas CA, Alsaker KV, Bonarius HPJ, Hendriksen WT, Yang H,Beamish JA, Paredes CJ, Papoutsakis ET. 2003. DNA array-based tran-scriptionalanalysisofasporogenous,nonsolventogenicClostridiumaceto-butylicum strains SKO1 and M5. J. Bacteriol. 185:4539 – 4547. http://dx.doi.org/10.1128/JB.185.15.4539-4547.2003.

46. Dürre P. 2005. Handbook on clostridia. Taylor & Francis, Boca Raton, FL.47. Fischer RJ, Helms J, Durre P. 1993. Cloning, sequencing, and molec-

ular analysis of the sol operon of Clostridium acetobutylicum, a chro-mosomal locus involved in solventogenesis. J. Bacteriol. 175:6959 –6969.

48. Petersen DJ, Cary JW, Vanderleyden J, Bennett GN. 1993. Sequence andarrangement of genes encoding enzymes of the acetone-production path-way of Clostridium acetobutylicum ATCC824. Gene 123:93–97. http://dx.doi.org/10.1016/0378-1119(93)90545-E.

49. Nair RV, Bennett GN, Papoutsakis ET. 1994. Molecular characterizationof an aldehyde/alcohol dehydrogenase gene from Clostridium acetobuty-licum ATCC-824. J. Bacteriol. 176:871– 885.

50. Nair RV, Papoutsakis ET. 1994. Expression of plasmid-encoded Aad inClostridium acetobutylicum M5 restores vigorous butanol production. J.Bacteriol. 176:5843–5846.

51. Jones SW. 2011. Elucidating the transcriptional regulation of sporu-lation in Clostridium acetobutylicum. Ph.D. thesis. Northwestern Uni-versity, Evanston, IL.

52. Tummala SB, Welker NE, Papoutsakis ET. 1999. Development andcharacterization of a gene expression reporter system for Clostridiumacetobutylicum ATCC 824. Appl. Environ. Microbiol. 65:3793–3799.

53. Cornillot E, Nair RV, Papoutsakis ET, Soucaille P. 1997. The genes forbutanol and acetone formation in Clostridium acetobutylicum ATCC 824reside on a large plasmid whose loss leads to degeneration of the strain. J.Bacteriol. 179:5442–5447.

54. Alsaker KV, Spitzer TR, Papoutsakis ET. 2004. Transcriptional analysisof spo0A overexpression in Clostridium acetobutylicum and its effect onthe cell’s response to butanol stress. J. Bacteriol. 186:1959 –1971. http://dx.doi.org/10.1128/JB.186.7.1959-1971.2004.

55. Takamatsu H, Chikahiro Y, Kodama T, Koide H, Kozuka S, TochikuboK, Watabe K. 1998. A spore coat protein, CotS, of Bacillus subtilis issynthesized under the regulation of sigma(K) and GerE during develop-ment and is located in the inner coat layer of spores. J. Bacteriol. 180:2968 –2974.

56. Takamatsu H, Kodama T, Imamura A, Asai K, Kobayashi K, NakayamaT, Ogasawara N, Watabe K. 2000. The Bacillus subtilis yabG gene istranscribed by SigK RNA polymerase during sporulation, and yabG mu-tant spores have altered coat protein composition. J. Bacteriol. 182:1883–1888. http://dx.doi.org/10.1128/JB.182.7.1883-1888.2000.

57. Santangelo JD, Kuhn A, Treuner-Lange A, Durre P. 1998. Sporulationand time course expression of sigma-factor homologous genes in Clos-tridium acetobutylicum. FEMS Microbiol. Lett. 161:157–164. http://dx.doi.org/10.1111/j.1574-6968.1998.tb12943.x.

58. Sebaihia M, Wren BW, Mullany P, Fairweather NF, Minton N, StablerR, Thomson NR, Roberts AP, Cerdeno-Tarrraga AM, Wang HW,Holden MTG, Wright A, Churcher C, Quail MA, Baker S, Bason N,Brooks K, Chillingworth T, Cronin A, Davis P, Dowd L, Fraser A,Feltwell T, Hance Z, Holroyd S, Jagels K, Moule S, Mungall K, Price C,Rabbinowitsch E, Sharp S, Simmonds M, Stevens K, Unwin L, Whit-head S, Dupuy B, Dougan G, Barrell B, Parkhill J. 2006. The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mo-saic genome. Nat. Genet. 38:779 –786. http://dx.doi.org/10.1038/ng1830.

Al-Hinai et al.

298 jb.asm.org Journal of Bacteriology

on April 29, 2016 by guest

http://jb.asm.org/

Dow

nloaded from

59. Veening JW, Smits WK, Kuipers OP. 2008. Bistability, epigenetics, andbet-hedging in bacteria. Annu. Rev. Microbiol. 62:193–210. http://dx.doi.org/10.1146/annurev.micro.62.081307.163002.

60. Veening JW, Stewart EJ, Berngruber TW, Taddei F, Kuipers OP,Hamoen LW. 2008. Bet-hedging and epigenetic inheritance in bacterialcell development. Proc. Natl. Acad. Sci. U. S. A. 105:4393– 4398. http://dx.doi.org/10.1073/pnas.0700463105.

61. Chary VK, Meloni M, Hilbert DW, Piggot PJ. 2005. Control of the expres-sion and compartmentalization of sigma(G) activity during sporulation of

Bacillus subtilis by regulators of sigma(F) and sigma(E). J. Bacteriol. 187:6832–6840. http://dx.doi.org/10.1128/JB.187.19.6832-6840.2005.

62. Tracy BP, Gaida SM, Papoutsakis ET. 2008. Development and applica-tion of flow-cytometric techniques for analyzing and sorting endospore-forming clostridia. Appl. Environ. Microbiol. 74:7497–7506. http://dx.doi.org/10.1128/AEM.01626-08.

63. Ferrell JE. 2002. Self-perpetuating states in signal transduction: positivefeedback, double-negative feedback and bistability. Curr. Opin. Cell Biol.14:140 –148. http://dx.doi.org/10.1016/S0955-0674(02)00314-9.

�K of Clostridium acetobutylicum Plays Dual Roles

January 2014 Volume 196 Number 2 jb.asm.org 299

on April 29, 2016 by guest

http://jb.asm.org/

Dow

nloaded from