Sporulation and Primary Sigma Factor Homologous Genes in Clostridium acetobutylicum


Using a PCR-based approach, we have cloned various cr factor homologous genes from Clostridium acetobutylicum DSM 792. The nucleotide sequence of the dnaE-sigA operon has been determined and predicts two genes encoding 69-and 43-kDa proteins. The deduced DnaE amino acid sequence has approximately 30%So amino acid identity with protein sequences of other primases. The putative sigA gene product shows high homology to primary r factors of various bacteria, most significantly to Bacillus subtilis and Staphylococcus aureus. Northern (RNA) blot analysis revealed that both genes form an operon, which is clearly expressed under conditions that allow for cell division. A promoter sequence with significant homology to the r -dependent Bacillus promoters preceded the determined transcriptional start point, 182 bp upstream of the GUG start codon of dnaE. The homologous genes to Bacilus spp. sporulation r factors G, E, and K have been cloned and sequenced. Indirect evidence for the existence of CFF was obtained by identification of a DNA sequence homologous to the respective Bacillus consensus promoter. Southern hybridization analysis indicated the presence of CrD and CH homologous genes in C. acetobutylicum. A new gene group conserved within the eubacteria, but with yet unspecified functions, is described. The data presented here provide strong evidence that at least some of the complex regulation features of sporulation in B. subtilis are conserved in C. 

acetobutylicum and possibly Clostridium spp. Sigma (ao) factors are transcription factors that operate global switches in gene expression by conferring promoter- specific transcription initiation on bacterial RNA polymerase. Many a factors, controlling the transcription from housekeeping promoters as well as several coordinately regulated sets of genes, have been identified in the last decade (for reviews, see references 30, 40, and 50). On the basis of sequence informa- tion, or factors seem to fall into two major classes, of which only the u70 homologous group will be considered here. A great deal of information has been obtained on the major vegetative u factors, including nucleotide sequences from various bacteria (40), conservation and evolution of the operon structure (72), as well as functional domains of the protein (30,40). Alterna- tive or factors are involved in regulation of many postexponen- tial-phase phenomena, such as sporulation in Bacillus spp. (20, 44), stationary phase in enterobacteria, and motility and chemotaxis in many bacteria. However, with the exception of Bacillus spp., only limited information is currently available on or factors in gram-positive organisms. Nucleotide sequences of vegetative cr factors have been determined only for Streptomyces spp. (36,67) and Staphylococcus aureus (5) in addition to a sporulation-specific a factor (WhiG) from Streptomyces coeli- color (12). 
The orchestral expression of stage-specific gene sets during sporulation in Bacillus subtilis, governed by specific a factors, has attracted much scientific interest (20,44). On the basis of morphological studies, sporulation in Bacillus spp. and sporu- lation in Clostridium spp. have long been suggested to employ similar mechanisms (34). Nevertheless, almost no attention has been paid to the genetic analysis of sporulation in the spore- forming, gram-positive, anaerobic genus Clostridium, which is of considerable biotechnological potential (77). Hitherto, sequence information of clostridial sporulation genes is available only for two ssp genes from Clostridium perfringens (31) and the spoIfD homolog of C. acetobutylicum (28). 
Among the more than 120 clostridial species, C. acetobutylicum, C. perfringens, C. botulinum, C. pasteurianum, and C. thermocellum have been studied at the genetic level (3,77,79,80). The first and second species are now accepted as model organisms of apathogenic and pathogenic clostridia, respec- tively (3,80). C. acetobutylicum is characterized by its meta- bolic ability to shift from a predominantly acidogenic to a solventogenic fermentation pattern (4,34). Along with the shift to solventogenesis, the cells undergo a series of morpho- logical and physiological changes in motility, shape, and granulose content. These accumulated data suggest that the solven- togenic phase is an important feature for sporulation in C. acetobutylicum but not a sporulation-specific event (33, 41-43, 49, 57), as has been shown for the solventogenic C. thermosac- charolyticum (39). 
An understanding of the developmentally regulated sporu- lation process and its impacts on product formation might be helpful for the development of an optimal fermentation pro- cess. In this report, we provide the first description of the molecular mechanisms involved in sporulation and transcriptional control mediated by a factors in Clostridium spp. 

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions. Chro- mosomal DNA of C. acetobutylicum DSM 792 (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) was used for recombinant DNA library construction and PCR amplifications. Total RNA of batch and continuous cultures was obtained from strain DSM 1731. Genomic DNA for comparative analysis of C. acetobu- tylicum strains was isolated from ATCC 824, DSM 1731, NCIMB 8052, and P262 (laboratory stocks). B. subtilis 168 (DSM 402) chromosomal DNA served as a control template in the PCR experiments. Escherichia coli JM83 (78) and SURE (Stratagene GmbH, Heidelberg, Germany) were used as hosts, and pUC9 (78) and pEcoR252 (kindly supplied by D. R. Woods, University of Cape Town, Cape Town, South Africa) were used as vectors for genomic library construction and cloning experiments. 
In batch culture, C. acetobutylicum was routinely grown under strictly anaerobic conditions at 370C in clostridial basal medium (51). The growth experiment in batch culture was done in CAMM medium, which has been described as being particularly useful for induction of sporulation (41). Continu- ous culture experiments with strain DSM 1731 were performed as described by Bahl et al. (2). E. coli was cultivated at 370C in LB medium (58) supplemented with ampicillin (50 pug/ml), isopropyl-13-D-thiogalactopyranoside (IPTG; 50 jig/ml), or 5-bromo-4-chloro-3-indolyl-,3-D-galactopyranoside (X-Gal; 40 pg/ml) as required. 
Physiological and morphological determinations. Numbers of total bacterial cells as well as percentages of motile cells, clostridial stages, forespores, and spores were determined in a counting chamber with a phase-contrast microscope (Standard RA; Carl Zeiss, Oberkochen, Germany). Product formation was measured by gas chromatography (model 439 gas chro- matograph; Packard Instruments GmbH, Frankfurt, Germany) as described by Bahl et al. (2). 
DNA isolation and manipulation. Chromosomal DNA of C. acetobutylicum and B. subtilis was isolated by the method of Marmur (47), with the modifications described by Bertram and Durre (6). For isolation of plasmids from E. coli, the Quiagen Midi kit (Diagen GmbH, Dusseldorf, Germany) was used. DNA was manipulated by standard methods (58) with restric- tion enzymes and T4 DNA ligase obtained from GIBCO/BRL GmbH (Eggenstein, Germany) according to the manufactur- er's instructions. PCR. PCR amplifications were performed in 100-pld volumes containing primers (0.5 ,uM each), deoxynucleoside triphos- phates (200 ,uM each), chromosomal DNA (< 0.5 ,ug), and Taq polymerase (2.5 U; GIBCO/BRL) in 50 mM KCl-10 mM Tris HCl (pH 7.5)-1.5 MgCl2-10 ,ug of gelatin, covered with light mineral oil. Temperature cycling was performed by a programmable thermocycler (Trio-Thermoblock; Biometra biomedizinische Analytik GmbH, Gottingen, Germany) with the following conditions: 94°C for 1 min, 37°C for 30 s, and 72°C for 1 min/1,000 bp for 35 cycles. The conditions were adjusted by raising the annealing temperature and lowering the primer concentrations if unspecific fragments were amplified. Synthetic oligonucleotides for PCR and sequencing reactions were prepared by a Gene Assembler Plus (Pharmacia LKB GmbH, Freiburg, Germany) as instructed by the manufacturer. The following heterologous primers (in 5'-to-3' direction) for PCR amplification of cr factor fragments were used (nucleoti- des at wobble positions are indicated in parentheses): for sigA, GGCTCGAGAT(ACT)GC(ACGT)AA(AG) (AC)(AG) (ACGT)TA (SA2.1), containing an XhoI linker at the 5' end, and GT(AGT)AT(ACGT)GC(CT)TG(ACGT)C(GT)(AGT) ATCCACC (SA2.3); for sigD, TTGGATCCTGGAA(AG)GA (AG)TGGAA(AG)GA (SD2.1), containing a BamHI linker at the 5' end, and AAGAATTCGC(AG)TA(AT)GT(AG)TC (AG)AA(CT)TT (SD2.3), containing an EcoRI linker at the 5' end; for sigE, TITGGATCCAA(AG)TT(CT)GA(AG)AA(CT) AC(ACGT)GG (SE2.1), containing a BamHI linker at the 5' end, and AAGAATTCCC(AG)TCCCA(AG)TC(AGT)AT (AG)TI' (SE2.3), containing an EcoRI linker at the 5' end; and for sigH, TTGGATCCAA(AG)TA(CT)(AC)G(ACGT)AA (CT)TT(CT)GT (SH2.1), containing a BamHI linker at the 5' end, and AAGAATTCTC(ACGT)GC(AG)AA(ACGT)GC (CT)TT(AG)AA (SH2.3), containing an EcoRI linker at the 5' end. These oligonucleotides correspond to the amino acid sequences of subregions 2.1 (core binding region) and 2.3 (RpoD box), known to be conserved in many a factors (30,40). Partial genomic libraries of C. acetobutylicum. Chromosomal DNA of strain DSM 792 was digested with HindIII and subjected to sucrose density centrifugation (10 to 40% [wt/ vol]). Fractions containing fragments of 0.8 to 3.5 kbp were dialyzed against TE buffer (10 mM Tris HCl [pH 8.0], 1 mM EDTA), ethanol precipitated, and ligated into HindIII-digested pUC9. Ligation mixtures were directly used for transformation of E. coli cells by means of electroporation with a Gene Pulser (Bio-Rad Laboratories GmbH, Munchen, Ger- many) as described by Dower et al. (17). Different libraries resulted from partial digestion of chromosomal DNA with Sau3AI and ligation into pEcoR252 or pUC9 (21, 22). Colo- nies from agar plates were transferred to Hybond N nylon filters (Amersham Buchler GmbH, Braunschweig, Germany), lysed with NaOH, washed as previously described (7), and subjected to the hybridization procedure described below. 
Southern hybridization. Chromosomal DNA of C. acetobu- tylicum was digested to completion with the appropriate re- striction enzymes, separated on 1% agarose gels, and transferred to nylon membranes (GeneScreen Plus; Dupont NEN Research Products, Dreieich, Germany) by capillary transfer in 10x SSC (1 x SSC is 0.15 M NaCl plus 15 mM sodium citrate). 
Prehybridization was performed for 1 to 3 h at 60°C in 10 ml of the following solution: 0.2% (wt/vol) polyvinylpyrrolidone, 0.2% (wtlvol) bovine serum albumin, 0.2% (wt/vol) Ficoll 400, 0.1% (wt/vol) sodium pyrophosphate, 10% (wt/vol) dextran sulfate, 1 M NaCl, 1% (wt/vol) sodium dodecyl sulfate (SDS), 50 mM Tris HCl (pH 7.5), and 100 ,ug of denatured salmon sperm DNA per ml. a-32P-labeled PCR-generated DNA probes were prepared as previously described (60) and denatured for 10 min at 95°C immediately prior to hybridization. Hybridiza- tion temperatures were 60°C for homologous and 40°C for heterologous DNA probes. Following hybridization for 10 to 15 h, the membranes were washed twice in 2x SSC and once in 2x SSC-1% (wt/vol) SDS for 5 min at room temperature and then subjected to autoradiography. DNA sequenning and sequence analysis. The nucleotide sequence was determined by primer walking with the dideoxy- chain termination method, using the T7 sequencing kit from Pharmacia LKB. All nucleotide data presented were confirmed by sequencing the opposite strand. The dideoxy-terminated fragments were separated on 55-cm wedge-shaped thickness gradient gels (0.2 to 0.4 mm, 6% [wtlvol] polyacrylamide) with a Macrophor sequencing unit (Pharmacia LKB) as recommended by the manufacturer. The computer-assisted analysis was done with the GeneWorks program (IntelliGenetics, Inc., Mountainview, Calif.) on a Macintosh Ilsi computer (Apple Computer, Inc., Cupertino, Calif.). Sequence comparison results were obtained by utilization of the Bestfit program (Genetics Computer Group Inc. sequence analysis software package, version 6.0 [14]) on a VAX 9000 computer. 
RNA isolation and analysis. Samples (8 ml each) of C. acetobutylicum cells were harvested from batch cultures at various time points, and total RNA was prepared by the hot phenol-chloroform method (52), with recently described mod- ifications (23). For concentration determinations, the optical density was measured at 260 nm (optical density of 1 corresponds to 40 jig/ml) in a model 601 spectrophotometer (Milton Roy, Rochester, N.Y.). Total RNA for Northern (RNA) blots was separated in denaturing formaldehyde agarose gels and transferred to nylon membranes (GeneScreen Plus) as de- scribed by Sambrook et al. (58). Fragment sizes were estimated by comparison with an RNA ladder (GIBCO/BRL). Hybrid- ization and washing were performed as described above for the Southern hybridization procedure. After the probes were stripped off by heating the membranes for 30 to 120 s at 95TC in 0.1 x SSC-0.1% SDS, Northern blots could be reused. Primer extension analysis was performed as described by Gerischer and Durre (23) except that SuperScript reverse transcriptase (GIBCO/BRL) was used. 
Nucleotide sequence accession number. The DNA se- quences reported in this paper have been submitted to EMBL and GenBank databases and assigned accession numbers Z23079 (sigEiG), Z23080 (dnaE-sigA), and L23317 (sigK). 

RESULTS

Cloning and sequencing of the dnaE-sigA operon from C. acetobutylicum. Sequence information from the highly con- served region 2 of the vegetative cr factor of B. subtifis (RpoD) (30, 40) was used for preparation of degenerate oligonucleotides. The oligonucleotides were employed as primers in a subsequent PCR amplification using genomic DNA from C. acetobutylicum DSM 792 as the template source, according to the approach of Engel and Ganem (18). A 156-bp PCR fragment was obtained with B. subtilis as well as C. acetobutylicum genomic DNA, subcloned, and partially sequenced. The deduced amino acid sequence of the clostridial fragment showed significant homology to RpoD of B. subtilis (24) and was therefore radiolabeled for screening a partially digested Sau3AI genomic library of C. acetobutylicum (21). A positive clone with a 4,435-bp insert was identified, and the respective plasmid was designated pSA21. The cloned fragment con- tained seven internal Sau3AI sites but was originally contigu- ous on the chromosome, as proved by Southern hybridization and PCR (59). 
Figure 1 shows 3,720 bp of the nucleotide sequence from the 4,435-bp insert and the deduced amino acid sequences of two large open reading frames. The first open reading frame exhibited significant homology to the dnaE(G) genes of enter- obacteria, Buchnera aphidicola, Rickettsia prowazekii, and B. subtilis (Table 1), encoding primases, and was designated accordingly. On the basis of amino acid sequence comparison of DNA primases from other organisms, we propose that C. acetobutylicum dnaE encodes a protein with a calculated mass of 69 kDa. All known DNA primases are 50 to 60% homolo- gous to each other, as judged by pairwise alignment, except for the enterobacterial primases, which exhibit 86% identity to each other. By means of sequence comparison, the second open reading frame was identified as the sigA homologous gene of C. acetobutylicum, presumably encoding the primary cr factor (Table 2). The putative clostridial SigA protein is of the smaller, B. subtilis-like type with a calculated molecular mass of 43 kDa. The overall similarity as determined by multiple sequence alignment was highest to the major vegetative ar factors of gram-positive bacteria, especially to the respective proteins of B. subtilis and S. aureus (data not shown). Highest variability between SigA from C. acetobutylicum and B. subtilis was found among the first 100 amino acids, whereas the sequences of regions 2.4 and 4.2 (30) were identical in both proteins. These two regions are believed to mediate recognition of -10 and -35 regions from cognate promoters (10, 16, 30), and certain amino acids within these regions are known to suppress mutations in specific promoter nucleotides (35). The helix-turn-helix motif of region 4.2 is known to be highly conserved exclusively among the primary v factors (40). 
A putative ribosome-binding site (5'-AGGAGG-3') is lo- cated 8 bp upstream of the sigA start codon. However, no such site was detected upstream of dnaE, which is in agreement with data reported for R prowazekii (46). Immediately downstream of sigA is a 10-bp inverted repeat with the potential to form a stable stem-loop structure (AG, -69 kJ/mol) (71) followed by a run of U's, resembling a rho-independent transcriptional terminator (13). Several possible, partly overlapping stem-loop structures are located between the GUG start codon of dnaE and the transcription start point, as discussed below. 
RNA analysis of the dnaE-sigA operon. During batch culti- vation of C. acetobutylicum DSM 1731 in CAMM medium, several morphological parameters, such as motility and ap- pearance of phase-bright swollen clostridial forms, proposed to be involved in sporulation and solventogenesis (33), were monitored in addition to the product formation pattern (Fig. 2). We conducted Northern blot experiments to investigate a possible operon structure of dnaE and sig4, as has been shown for many gram-negative bacteria as well as B. subtilis (72). Total RNA was isolated from cells harvested at different time points during the CAMM batch culture and from acidogenic and solventogenic continuous cultures of C. acetobutylicum DSM 1731. Three signals (3.0, 2.2, and 1.2 kb) were observed with a PCR-generated radiolabeled DNA probe, complemen- tary to region 2 of the clostridial sigA gene (positions 2917 to 3192) (Fig. 3). In continuous culture under acidogenic (Fig. 3) as well as solventogenic conditions (data not shown), only the 3.0-kb transcript was detected. With RNA from E. coli con- taining pSA21, a slightly smaller transcript of approximately 2.8 kb was detected, suggesting that the clostridial dnaE-sigA operon is transcribed from a different promoter in E. coli. 
RNA harvested from overnight cultures of C. acetobutylicum prior and after a heat shock (30 to 42°C for 10 min) showed identical hybridization signals with the sigA gene probe (Fig. 

3).

A transcription start point of the dnaE-sigA operon was determined by primer extension analysis, using oligonucleo- tides cr4336 (5'-ATAAGACATTCCACCCT-3') and c4342 (5'-AACAACTAAAJTCCCAT-3'), complementary to the 5' region of dnaE (Fig. 4). A single transcriptional start site, obtained with both oligonucleotides, was mapped to position 438, 182 bp upstream of the dnaE start codon (Fig. 1). Additional signals appeared with both oligonucleotides, repre- senting most likely nonspecific break-off products of the re- verse transcriptase reaction, since they differed in size. Preced- ing the mapped transcription start point, a sequence (5'-AAGGATTITA-12 bp-GAATTAA-4 bp-transcription start point-3') with significant homology to the arH-dependent con- sensus promoter ofB. subtilis (68), showing only one mismatch, was identified. Known C. acetobutylicum promoter sequences (21, 23, 61, 73) could not be detected within the appropriate distance. RNA from batch culture cells yielded weaker signals with fewer nonspecific bands, possibly because only 3 ,ug of RNA was applied, as opposed to 10 pg from continuous culture cells. The 17-bp prolonged RNA transcript from the 46-h batch culture cells, obtained with oligonucleotide cr4342, is most likely an artifact, since it was detected in only one case. 
No signals were obtained with RNA isolated from batch culture cells older than 60 h. Attempts to identify an internal transcription start point upstream of sigA4 by primer extension, CCATCAATCCAGGGCATAAG TATAAAAATCATTCCTGMT ATATTACAAAACGTGAGTTA TCAAGTGTAGGTAGAATTCT TGTTACATTAATGCCGTGCC TAAAAAACCATTCAGTAGTT 120 GAATATACAAATAGAAGCTC TCCTTTATCATAGTATACTT TTTCAAGCAGTATTTTTAGA ACCTAAAGTTAGTTTATACA CAGCCCTTTGTTTTTCAGTA TCTTTAAATTTAATTTGTAA 240 TATTTCCGCATCTTGAAGAT TATATTTTGGTAATATATAT TT7TTTATTGAATCTCCGGA AGAAGAATATGTGAGGGATT AATAAAAAACACCATTGAAT GGGTAAACTAAGAGTATGAA TAGAATAATTTTATTATTTT TTAAAATAAAATTICTCAAA AGAAGGATTTAAACTTTTTT TGTCGAAATTAATTATGCTC 600 ATGGGAATTTAGTTGTTAGG GTGGAATGTCTTATTATAGC TGAAGATATAATTCAAAAAG TAAAAGATTTAAATGATATA GTGGATATTGTATCTGAAAA GGTAAAGCTTAAACGTACAG 720 which could give rise to the 1.2-kb transcript, failed. Even though the clostridial dnaE-sigA operon is highly expressed in E. coli (Fig. 3), we were unable to obtain primer extension signals with the above-mentioned oligonucleotides and RNA from the respective E. coli clone. This fact further strengthens the implication that a different promoter is utilized for expres- sion of the cloned operon in E. coli. 
aZ M E C L I I A E D I I Q K V K D L N D I V D I V S E K V K L K R T G GACGTAATTATTCGGGGCTT TGCCCATTTCATCATGAAAA AACACCGTCATTTAGTGTAT CTCAGGATAAACAAATATAT AAATGCTTTGGATGTGGAGA AGCAGGAAATGTTATAACTT 840 R N Y S G L C P F H H E K T P S F S V S Q D K Q I Y K C F G C G E A G N V I T F TTGTAATGAAGACTAAAAAC TTFTClTTTTAGAGGCGGT AAAATACCTTGCGGACAAAG TTAACATTACTATAGACGAT AATAAAGATAAGTACTATGC AAAAAATAAAGAGAAATATG 960 V M K T K N F S F V E A V K Y L A D K V N I T I D D N K D K Y Y A K N K E K Y D ACAAATTGTATAGTATGAAT GTTGAAGCAGCCAGGTATTT TT7TAGAAATAGATTCTA ATTCAAAAGCAAAAAAATAT TTTTTGGATAGAGGAATTAA TCAAAAACCATGAGAAGGT K L Y S M N V E A A R Y F F R N L D S N S K A K K Y F L D R G I N Q K T M R R F TTGGACTTGGATATGCTAAG GATGACTGGAGACAGCTTCT AAGTTTTATGAAATCTAAGG GGTATACAGAATTAGAACTT ATTGAAGCAGGACTTATAGT ACAGAAAAATAAAGCTGTCT G L G Y A K D D W R Q L L S F M K S K G Y T E L E L I E A G L I V Q K N K A V Y ATGATAGGTTTCGAAATAGG GTTATATTTCCTGTATTTGA CTATAAAGGCAAGGTTATAG GGTTT'GAGGTAGAGTTTTA GATGATTCTAAACCAAAATA TrTAAACTCTCCAGAAACCA D R F R N R V I F P V F D Y K G K V I G F G G R V L D D S K P K Y L N S P E T K AGATATITAAAAAAGGAACT AACCTTrATGGACTAAATTT TGCAGTGAAAAATAATATAG GGGATACAATTATAATTGTA GAGGGATACATGGATTGTAT ATCTCTTCATCAATATGGTA I F K K G T N L Y G L N F A V K N N I G D T I I I V E G Y M D C I S L H Q Y G I TAAATAACGTTGTGGCTTCG CTTGGAACAGCACTTACGAA AGATCAGGCAAAGTTGTTAA AGAGATATGCTAGTAAAGTG GTAATATCATATGACGCTGA TACGGCTGGTCAGGCTGCTA N N V V A S L G T A L T K D Q A K L L K R Y A S K V V I S Y D A D T A G Q A A T CACTTAGAGGGTTTGATATA TTAAGAGAAGCAGGATTTGA TATAAAAATAATAAAAATCC CAGATGGTAAAGATCCAGAT GAATTTGTGAGAAAAAATGG TAAAGAGGCTTTTATGAGGC L R G F D I L R E A G F D I K I I K I P D G K D P D E F V R K N G K E A F M R L TTGTAGAGAATGCTGTITCA ATTATAGACTACAGGATAAA TAGGGCCTTTGAAAACGTCA ATTTGAAGGATAGCGAAAGT GTTATAAAATATGTTAAGGA TATTTCGGAAATAATCGAAG V E N A V S I I D Y R I N R A F E N V N L K D S E S V I K Y V K D I S E I I E E AGTTAGATCCAGTAGAGAAA GATGTCTATATCAAGAAAAT TTCAGATAAAACGGGAATAA GAGAACAAGCAATTTATGAT TTGATTAGTACTGATATTCA GAAAAGTGGCAATAACATTG L D P V E K D V Y I K K I D K T G I R E Q A I Y D L I S T D I Q K S G N N I E AAAAAATGAATAAAAATAGC AATTATGGACAAAATTTATA TGTGGAATCCGCACACGTTA AATCCGAAAGATATTTACTT AGATTAATGATTGAACATAA AGATATACGCAGTTATATAG 0 K M N K N S N Y G Q N L Y V E S A H V K S E R Y L L R L M I E H K D I R S Y I E AGTCCAAAATTAGTGCGGAC GATTTAATAATGGAAAGCCA TAAAAAAATATATAACATTA TATTGAATTTTGAAGATAAT TTAAGTAGTGATCTAAAATC TAAATTTGGTTTTATTGAAT S K I S A D D L I M E S H K K I Y N I I L N F E D N L S S D L K S K F G F I E S CAAGATGTGATGATGGGGAA AGTTCCTCTGAAATTGTAAA GATAGAAGAACAGAATATAA TGCTTTCAGAAAACAATGTT AAAGATCTTGTAGATGATTT TATTACAAACATTAAAAAAT R C D D G E S S S E I V K I E E Q N I M L S E N N V K D L V D D F I T N I K K F TTAGATTAGAGGAGTCAAAA AAGAAGATTATGAGTGAAAT TAAAGGCCTTGAAGAAAAGG GATTGTTTGATGAGACCATT GAACTTATCAAAAGAATGGA AGAAATACAAAATCAGTTAG R L E E S K K K I M S E I K G L E E K G L F D E T I E L I K R M E E I Q N Q L D ACAATCTATAGAATAACGGA TGATG GTAATGAA AAATAAGACTGAAGTTAAAA ACGGGGGAGAAAAGAAAAAC TCAAAAAAAGTGTCAAAAGA AGAAAGCGCAAAAGAAAAAA N L OD 2gA M K N K T E V K N G G E K K N S K K V S K E E S A K E K N ATGAAAAAATGAAAATTGTC AAAAACTTAATAGATAAAGG GAAAAAGAGTCGAAGTtTAA CGTATAAAGAAATAATGGAT GAACTTCAGGAAGTTGACCT AGGACCAGAGCAGATAGAAA E K M K I V K N L I D K G K K S G S L T Y K E I M D E L Q E V D L G P E Q I E K AGATATATGAGGTCVTTGAA TCTGTTGGCGTTGATGTAGT AGGTGACATGCATGAAATAG AGGTTGAAGAAGAAGATTTA GATTTGACAATACCCGAGGG AATAGCTATAGATGACCCTG I Y E V L E S V G V D V V G D M H E I E V E E E D L D L T I P E G I A I D D P V TAAGAATGTACCTTAAAGAA ATTGGTAAAGTTCCGTTGTT ATCACCAGAGGAAGAAATTG ATITAGCGCAAAGAATTAAA AACGGTGATAGATCAGCAAG GAAGAAGCTGCAGAAGCTA R M Y L K E I G K V P L L S P E E E I D L A Q R I K N G D R S A R K K L A E A N ACTTAAGACTTGTTGTAAGT ATPGCAAAAAGATATGTTGG AAGAGGGATGCTITTCCTG ATTTAATCCAGGAAGGAAAT TTAGGACTCATTAAGGCAGT AGAAAAGTTCGATTTCAAAA 3000 L R L V V S I A K R Y V G R G M L F L D L I Q E G N L G L I K A V E K F D F K K AAGGATTTAAATTTAGTACT TACGCTACTTGGTGGATAAG GCAGGCATAACTAGAGCTA TAGCGGATCAAGCAAGAACT ATAAGAATTCCGGTTCATAT GGTTGAAACCATAAATAAAC 3120 G F K F S T Y A T W W I R Q A I T R A I A D Q A R T I R I P V H M V E T I N K L TTATAAGAGTCTCAAGACAG TTGCTTCAGGAATTAGGTAG AGAACCTCAGCCAGAAGAAA TCGCTAAAATAATGGATATG CCGGTTGATAAGGTAAGAGA GATAATGAAGATAGCACAAG 3240 I R V S R Q L L Q E L G R E P Q P E E I A K I M D M P V D K V R E I M K I A Q E AGCCAGTATCACTTGAAACA CCTATAGGAGAAGAAGAAGA TAGTCATCTTGGTGATTTTA TTCCAGATGATGAAGCTCCT GCTCCAGCAGATGCTGCTGC GTTTAGAATGCTTAAAGAGC 3360 P V S L E T P I G E E E D S H L G D F I P D D E A P A P A D A A A F R M L K E Q AACTCCTAAAAATTCTAAAT ACATTAACTCCTAGGGAAGA GAAAGTTTTAAGACTTAGAT TTGGTCTCGATGATGGAAGA GCTAGAACACTTGAAGAGGT TGGAAAAGAGTTTAATGTTA 3480 L L K I L N T L T P R E E K V L R L R F G L D D G R A R T L E E V G K E F N V T CAAGAGAGAGAATAAGGCAG ATAGAAGCAAAAGCTCTTAG AAAACTAAGACATCCAAGTA GAAGCAAAAAATTAAAAGAT TATCTTGATTAATAAATACT AAGGTTATCCTTAATGGGTG 3600 R E R I R Q I E A K A L R K L R H P S R S K K L K D Y L
Cloning and sequencing of the sigE and sigG genes from C. 
acetobutylicum. Degenerate oligonucleotides, complementary to region 2 of the B. subtilis sporulation u factor E (30,40), were employed as primers in a PCR amplification using genomic DNA of C. acetobutylicum DSM 792 as a template. A 235-bp DNA fragment was amplified and subsequently radiolabeled for screening a HindIII partial gene bank of C. acetobutylicum DSM 792. The nucleotide sequence of a single positive clone (pSE1) with a 3,450-bp HindIII insert was determined, revealing six open reading frames (Fig. 5), the first of which was truncated and showed significant homology to crE of Bacillus spp. A probe consisting of the first 159 bp of pSE1 was then used to screen two different Sau3AI partial genomic libraries of C. acetobutylicum (21,22). In the second library, a positive clone (pT22) with a 2,416-bp Sau3AI insert could be detected. Its nucleotide sequence was determined and found to encode three open reading frames (Fig. 5). Computer-assisted sequence comparison for the putative products of the first three open reading frames of the complete DNA region showed high similarity to the Bacillus spp. sporulation-specific protease SpoIIGA and the sporulation or factors E and G (Table 3). Therefore, these genes were classified as the C. acetobutylicum homologous genes and designated spoHIGA, sigE, and sigG, accordingly. The respective DNA region and the deduced amino acid sequence is presented in Fig. 6. On the basis of homology to aG from Bacillus spp., the translation of the clostridial sigG starts at a GTG codon. The conservation of methionine residues in the sequence context of the a-helical segment in region 2.4 is striking, as this is known to suppress mutations in the promoter -13 and -12 positions of B. subtilis (15). A possible stem-loop structure (AG, -30 kJ/mol [71]) without the features of a prokaryotic rho-independent transcriptional terminator (13) was identified between sigE and sigG. Overlapping this possible secondary structure is an identifiable promoter sequence (5'-AGTATA-15 bp-GG- AATAAT-3') with reasonable homology to the uF consensus sequence (66), which is known to control expression of sigG in B. subtilis (20). Ribosome-binding sites could be identified upstream of the spoIIGA (5'-AGGTGG-3'), sigE (5'-GGGGGG-3'), and sigG (5'-AGGGGC-3') genes (Fig. 6). The putative transcript of sigG has a potential of forming a strong stem-loop structure (AG, -82.4 kJ/mol [71]), again lacking typical features of a rho-independent terminator. The deduced amino acid sequence of the spoIIGA gene product showed considerably lower homology to its Bacillus analog than the or factors (Table 3). However, the hydropathy profile of the B. subtilis SpoIIGA (63) was almost identical to that of the clostridial SpoTIGA (data not shown). 
The deduced amino acid sequence derived from open reading frame V exhibited significant similarity (46 to 59% identity) to three hypothetical bacterial proteins of equal size FIG. 3. Northern hybridization with the radioactively labeled PCR- generated fragment complementary to sigA. The lanes marked 12h to 90h contained 10 pLg each of total RNA isolated from C. acetobutylicum DSM 1731 at different time points during a batch cultivation in CAMM medium (Fig. 2). Total RNA (10 ,ug) for the next two lanes was isolated from strain DSM 792 prior (30'C) and after (HS) a heat shock from 30 to 420C for 10 min in batch culture. The lane marked pSA21 contained 10 ,ug of total RNA isolated from the E. coli clone with plasmid pSA21. In lane A, 7 ,ug of total RNA of an acidogenic continuous culture of C. acetobutylicum DSM 1731 was applied. Transcript sizes, as judged by comparison with size markers, are indicated at the left. 
with unknown function (Fig. 7). Particularly striking is a run of four arginines at the 5' end, found in the putative products of all of these completely sequenced genes. The respective open reading frames have been found in all cases in the vicinity of genes involved in DNA-RNA-protein interaction: nusB gene region of E. coli (antitermination factor) (69), dnaB gene of B. subtilis (involved in replication) (32), 16S rRNA region of Thermus thermophilus (translation) (29), and sigE-sigG gene region of C. acetobutylicum (regulation of sporulation). The conserved sequence of these proteins among phylogenetically distinct bacteria indicates an important and similar function within the eubacteria. No significant homologies were detected for any of the other open reading frames encoded in the sequenced DNA region. 
Cloning and sequencing of the sigK gene from C. acetobutylicum. A 159-bp PCR-generated DNA fragment (positions 971 to 1129; Fig. 6) was originally designed for detection of sigE in a partially Sau3AI-digested genomic library of C. acetobutylicum DSM 792 in vector pEcoR252 (21). A positive clone (pT4) that carried an approximately 4,350-bp insert was identified. However, sequencing of the first 900 bp of this insert revealed that pT4 did not contain the sigE gene but contained a gene homologous to crK of Bacillus spp. Only the 3' end of the respective open reading frame was encoded on the Sau3AI fragment. A 372-bp probe (positions 276 to 647; Fig. 8) was then used to screen a different, partially Sau3AI-digested genomic library of C. acetobutylicum DSM 792 in pUC9 (22). A positive clone (pT41) that carried an approximately 7-kbp insert was found. The nucleotide sequence (960 bp) of the DNA region encoding the sigK gene on this insert was determined, and computer-assisted analysis revealed an open reading frame encoding the putative a factor K (Fig. 8). On the basis of homology to CK from Bacillus spp., the translation of the clostridial sigK starts at a GTG codon. A ribosome-binding site matching the consensus sequence (5'-AGGAGG-3') was identified 8 to 13 bp upstream of sigK (Fig. 8). The deduced amino acid sequence similarity between uK from C. acetobu- tylicum, B. subtilis, and B. thuringiensis is shown in Table 3 sigA operon by primer extension analysis. Oligonucleotides a4336 (a) and u4342 (b), complementary to the 5' region of dnaE, were annealed to 10 ,ug of total RNA from acidogenic (lanes Ac) and solventogenic (lanes S) continuous culture and 3 ,ug of batch culture (Fig. 2) C acetobutylicum DSM 1731 cells. The primer-extended products were coelectrophoresed with a sequencing ladder generated with U4336 as the primer. The arrows on each side indicate the positions of the products obtained with both primers. similarity was slightly higher to the respective protein of B. thuringiensis, as in the case of CG. As in B. thuringiensis (1), the sigK gene of C. acetobutylicum was not interrupted by insertion of an unrelated DNA segment as known for sigK of B. subtilis (65). Two possible stem-loop structures, both lacking homol- ogy to rho-independent transcriptional terminators, were de- tected 19 and 108 bp downstream of the stop codon (13). 
Southern blot analysis of cr factor genes in various C. acetobutylicum strains. Earlier reports showed major differ- ences between C. acetobutylicum strains (61, 76, 80). There- fore, possible sequence variations of cr factor genes among these strains were checked by Southern hybridizations. The sigA probe used was complementary to parts of region 2 (positions 2917 to 3192; Fig. 1). The probe for sigE detection was identical to the PCR-generated fragment used for screen- ing of the HindIII genomic library (positions 1049 to 1267; Fig. 6). Both probes were obtained by PCR amplification with genomic DNA of strain DSM 792 as the template. With the sigE-specific probe, fragments of identical size were detected in strains DSM 792 and ATCC 824, whereas genomic DNA of P262 and NCIMB 8052 gave only very weak signals of different size, possibly due to unspecific hybridization (data not shown). 
Essentially the same result was obtained with the sigA-specific probe. Southern hybridization revealed only a single signal, indistinguishable in size and intensity, with genomic DNA of strains ATCC 824, DSM 792, and DSM 1731, suggesting that sigA is the only gene encoding a primary cr factor. However, P262 and NCIB 8052 gave much weaker signals, which in case of NCIMB 8052 was of different size, indicating again a genetic difference, even in this conserved gene. The signal intensity for the latter two strains was approximately equal in intensity to control DNA signals from B. subtilis and C. butyricum. These results are in good agreement with earlier observations (61,76,80), providing strong evidence for a specific grouping of different C. acetobutylicum strains. 
In addition to the above-mentioned a factors, we looked for homologous genes to the B. subtilis a factors H and D. 
Applying the approach of Engel and Ganem (18), we were unable to obtain specific PCR fragments with clostridial DNA and degenerate primers (see Materials and Methods). How- ever, when genomic DNA of B. subtilis was used, PCR fragments of the expected size were obtained, subsequently radiolabeled, and employed for Southern hybridization. In both cases, single signals were obtained in the hybridization with chromosomal DNA of C. acetobutylicum DSM 792. The size of these signals was distinct from those of the other a factors, thus indicating the presence of aH and aD homologous genes in C. acetobutylicum. 

DISCUSSION

This report presents the first analysis on a factor genes in the gram-positive genus Clostridium. The operon encoding the putative principal a factor and primase of C. acetobutylicum was cloned and sequenced. The dnaE gene is specified by an 1.8-kbp open reading frame, whose putative product is similar to other primases, and we therefore conclude that the cloned gene encodes the primase of C. acetobutylicum. The putative product of the sigA gene exhibits significant homology to primary a factors of other bacteria and is of the smaller, gram-positive type. Amino acid sequence identity of the a helix in region 2.4 and the helix-turn-helix motif in region 4.2 between C. acetobutylicum and B. subtilis, believed to mediate recognition of -10 and -35 regions from cognate promoters (10,30), provides strong evidence for identical -10 and -35 consensus promoter regions in both organisms, as was sug- gested earlier by sequence comparison of promoter regions (21,23,59,61,73). The predicted molecular mass for the clostridial a factor A of 43 kDa is in good agreement with the value of 46 kDa calculated from the migration of the protein in SDS-polyacrylamide gels (54). 
On the basis of Northern blot analysis, the two genes were shown to form an operon, which, however, is less complex than the analogous operons of gram-negative bacteria and B. subtilis (72), since it comprises only two genes. In contrast to its counterpart in E. coli (70), our experiments could not demon- strate any increase of sigA-specific mRNA under conditions of heat shock. However, no 3.0-kbp transcript was detected in this case; therefore, the presence of a heat-inducible promoter cannot be completely ruled out. The transcriptional start point of the dnaE-sigA operon was mapped 182 bp upstream of the dnaE start codon. This site was not preceded by a typical aA-dependent promoter structure; thus, a different promoter must have been utilized by E. coli for transcription of the cloned operon. However, we cannot rule out the possibility that a closer transcription start point exists, since the primers used were complementary to the sequence directly upstream of dnaE, including the first 14 nucleotides of the structural gene. Transcription of this operon may be regulated during different growth phases in C. acetobutylicum. In batch culture, the mRNA transcript comprising both genes was observed only in a Similarity data were derived from pairwise alignments of the respective amino acid sequences. The symbol comparison table of Gribskov and Burgess (25) was used to calculate percentage identity and similarity. 
TTTAAGAPGAGAAA ATGGTTATTTATTTGGATGT TTTAAT=TTTTAAAATTCAA TAGTAAATACTTTTCTATTG TACATAACAGCTCAAACTTT AAGAATAAAGGTTAAAATGA 120 
SD qOtII M V I Y L D V L I F E N S I V N T F L L Y I T A Q T L R I K V K M R GATATTTAATCCTTGCTGGA ATATTTGGAGGGCTTTACGT AATAGTTTTAGTTATTCCTA CACTTAAGATATTTTCATCT TTGATATTTAAAATAATAGC G AATGATAATTA 240 Y L I L A G I F G G L Y V I V L V I P T L K I F S S L I F K I I A A F L M I I I TATGCTTTAGGAAAAAAAGT TTAAGATTTAATATAAAAGC GCTGGCTGTACTATTATGT ATTCTATGGTTACAGCTGGT TTATGTTTCTTTATTGAATT AAATAATACAAGAGGAAGCT 360 C F R K K S L R F N I K A L A V L I M Y S N V T A G L C F F I E L N N T R G S Y
ATTTTAACGCCTTCATTGGT AATGTATCTTACAAGTGGAT mTAATAGCTATTATGATTA TATATATGTTTGTAAATAGA ATTATATGGTTCATAAACGA TAGAAAATTAACACAAAGTC 480 
F N A F I G N V S Y K W I L I A I M I I Y M F V N R I I W F I N D R K L T Q S L
TAATTTATGAAATAGAAATA TGTTTTAAGGATAATAGCAA ATTTATAAATGCC ¶TTTTGG ATACGGGCAATGAGCTTAGA GAGCCTATTACAAACTTACC TGTTATTGTTGTTGAAAAAG 600 
I Y E I E I C F K D N S K F I N A F L D T G N E L R E P I T N L P V I V V E K D ACATGGTAAGCGGTATAAAG TGGGATGATTGTCCAAAGTT TTATGTTCCATTTAGATTAT TTAATGGAAAGGCAGGTAAC CTTGAAGCTTTTAAACCTTC TTATGTAAAAATATACATAG 720 M V S G I K W D D C P K F Y V P F R L F N G K A G N L E A F K P S Y V K I Y I G GTGATAAAGTGGAAGTAAGA AATGCAATTATAGCTTTAAT AGACAATAAATTAAGCAGCT TAAATGATTATAATGCACTC TTATCTAGAGGAAGTATTTA AAA7IgT ¶TTATGAA 840 D K V E V R N A I I A L I D N K L S S L N D Y N A L L S R G S I SD J M K GTTTTTATTGCGCCTTTAA GTAAATTATTGTGTAGAATT AAATTGTFTMAAACAGACT GTACTATATAGGTGGAAATG ATGCGCTTCCGCCTCCTCTT TCAAAGGATGAAGAAGATGA 960 F L L R L L S K L L C R I K L F S N R L Y Y I G G N D A L P P P L S K D E E D D
TTTGGTAAACAAGCTTGTAA ATGGAGATGAGAGTATACGC TCCATACTTATAGAAAGAAA TTTAAGACTTGTAGTGTATA TTGCAAGAAAATTTGAAAAT ACAGGGGTTGGGGTTGAAGA 1080 
L V N K L V N G D E S I R S I L I E R N L R L V V Y I A R K F E N T G V G V E D CTTGA7TTTCAGTTGGAACCA TAGGGCTTATTAAAGCTGTA AATACCTTTGATCCAACCAA AAAAATAAAGTTAGCAACAT ATGGTTCAAGATGTATTGAA AATGAGATACTTATGTATTT 1200 L I S V G T I G L I K A V N T F D P T K K I K L A T Y G S R C I E N E I L M Y L AAGAAGAAATAGTAAAGTAA AAGCTGAAATTTCATTrTAT GAACCACTCAATATAGACTG GGATGGAAACAAGCCGCTTT TATCAGATATATTAGGTACT GATAATGATTGTGTATATAA 1320 R R N S K V K A E I S F Y E P L N I D W D G N K P L L S D I L G T D N D C V Y N TCTTATAGAAGGAGAGGTTG ATAAACAGCTTTTGTTATmT GCTCTAAAAAGTTAAACGA AAGAGAAAAAAGAATAGTTG AATTAAGATATGGACTTACA GGCGTTGGAGAAAAAACTCA 1440 L I E G E V D K Q L L L F A L K K L N E R E K R I V E L R Y G L T G V G E K T Q AAAGGAAGTTGCAGATATGC TGGGTATATCTCAATCCTAT ATTTCAAGGCTTGAAAAGAG AATAATTAAGAGATTAAAAA AAGAAATAAATAAAATGTTG TGAGTTTGTCTTTAGTATAA 1560 K E V A D M L G I S Q S Y I S R L E K R I I K R L K K E I N K M L AATAGCTCCCCTGAGGGAAT AATTTTAATGCTATTATTT CAP~~ATAAAGTGG TTATAAACAAGGTGAAATT TGCGGCGTTAATACATCTAA ACTTCCTGTTCTAAAAGAAA 1680 >>>> <<<< 8D aiG M V I N K V E I C G V N T S K L P V L K E K AAGAAATGCGTGAACT'NTA ATCAGTATGAGGAATGGAAA CACAACTGCCAGAGAAAAAT TCATAAAAGGAAATCTGAGA TTAGTACTTAGCGTTATCCA GAGGTTCAATAATAGAGGTG 1800 E N R E L L I S M R N G N T T A R E K F I K G N L R L V L S V I Q R F N N R G E AGAATGCAGATGATPTATTT CAGGTTGGGTGTATAGGGCT AATAAAGTCCATAGATAACT TTGATTTAAGTCAGAATGTA AAATTTTCTACTTATGCGGT TCCGATGATTATAGGGGAGA 1920 N A D D L F Q V G C I G L I K S I D N F D L S Q N V K F S T Y A V P M I I G E I TAAGGAGATAT ¶NAAGAGAC AATAACTCAATTAGAGTAAG TAGAWTCTAGAGACATAG CATACAGAGCACTACAAGTC AGAGATAGACTTATAAGTAA AAACAATAAAGAACCCACTG 2040 R R Y L R D N N S I R V S R S L R D I A Y R A L Q V R D R L I S K N N K E P T V
TTTCACAAATTGCAAAGGAA TTAAAAATACCTCGCGAAGA AGTGATTTTTGCCTTAGATG CTATTCAAGATCCCATATCG TTATTTGAACCAATATACCA TGACGATGGTGATGCAATAT 2160 
S Q I A K E L K I P R E E V I F A L D A I Q D P I S L F E P I Y H D D G D A I Y ATGTAATGGATCAAATAAGC GATAATAAAAATTTAGATGA CAGCTGGCTTCAAAATATAT CAATTAAAGAGGCTATGAAA AAGCTAAGCGATAGAGAAAA AATGATACTTAATATGAGGT 2280 V M D Q I S D N K N L D D S W L Q N I S I K E A M K K L S D R E K M I L N M R F TTTTTGATGGTAGAACACAA ATGGAAGTTGCAGATGAAAT AGGAATTTCACAAGCACAGG TATCTAGACTCGAAAAAACA GCACTGAAACATATGAAAAA ATATGTTTAATTAAATTAGG 2400 F D G R T Q M E V A D E I G I S Q A Q V S R L E K T A L K H M K K Y V
ATAAAAAATTGATCTACTTA TTAGAAGGGCTTCCAAAGGT TTTAATGGAAGCCCTATATA CATATTAGAAACAATAGAAT TTATTTAGAATTTTTTGCAT AAGGTAAGCATATTTTTATA 2520 FIG. 6. Nucleotide sequence of the spoIIGA-sigE-sigG DNA region. The predicted amino acid sequences for the three genes are shown below the DNA sequence. Potential stem-loop structures are marked by arrowheads below the sequence. The sequence marked by the bars above the nucleotides exhibits significant homology to uF-dependent promoters of Bacillus spp. Putative ribosome-binding (Shine-Dalgarno [SD]) sites are boxed. exponential and early postexponential growth phases, while two smaller transcripts, possibly encoding sigA only, were detected throughout all growth phases. In continuous cultures, however, the longer transcript was exclusively detected. This could indicate a permanent expression of sigA, while transcription of the dnaE gene, specifying the replication protein primase, would occur only during growth phases that are characterized by cell division. On the other hand, we cannot exclude the possibility that the two shorter transcripts are the result of less specific hybridization, since no respective tran- scription start points could be detected in the primer extension experiments at the time points when only these transcripts were found. The lack of a suitable ribosome-binding site preceding dnaE might be a regulatory feature in control of primase protein levels, as was also suggested for E. coli (45). A transcript of the dnaE-sigA operon has the potential of forming several stem-loop structures in its 5' noncoding region, which might serve three possible functions, (i) stabilization of the 3.0-kbp transcript, (ii) binding site for endoribonuclease at- tack, or (iii) inhibition of RNA polymerase read-through, and thus serve as a means of regulation. The identification of a aH-dependent promoter upstream of dnaE in C. acetobutylicum reveals similarities to the transcriptional regulation of the analogous operon in B. subtilis, in which three cH-dependent promoters in addition to two &k-dependent promoters have been shown to participate in initiation of transcription (11,55,56,75). The Southern hybridization data for sigA, its sequence homology to other primary a factors, and the similar operon structure support the hypothesis that sigA encodes the major vegetative ( factor of C. acetobutylicum. 
In B. subtilis, four sporulation-specific r factors play crucial regulatory roles within this developmental process. The two early sigma factors CrE and aF determine the establishment of a distinct cell-specific program of gene expression, whereas aG and uK are synthesized at later stages in the prespore com- partment and the mother cell, respectively (20,44). In this paper, we reported the cloning and sequencing of homologous genes for three of these u factors from C. acetobutylicum. The --------------------------------_---- identification of a DNA sequence upstream of sigG, with homology to a'e-specific consensus promoters of Bacillus spp. 

Ca ----------------------MAIRRRRE CLECSKRYTT YEKVEDIPIL


Tt ----------------------MAIRRRRE CPSCGRRFTT YERTQLEPLM BS

(66), provides indirect evidence for the existence of the fourth af factor in C. acetobutylicum, cF. The sequential arrangement of the genes encoding SpoIIGA, arE, and aoG is conserved between Bacillus spp. and C. acetobutylicum, with the interest- ing feature that the former or factor is active only in the mother cell during early sporulation, while the latter is synthesized specifically in the prespore compartment at later stages (20,44). The conservation of this arrangement might indicate a yet unknown selective advantage. The sigE gene of C. acetobutylicum is the first gene known to be terminated by an UGA stop codon in this organism. Another sequenced sporulation-specific gene of C. acetobutylicum, spoIID (28), known to be under transcriptional control of CE in B. subtilis, is preceded by a sequence with homology to the B. subtilis consensus sequence for UE-dependent promoters (15). This fact together with the conservation of region 2.4, presumed to specify -10 promoter region binding (15), provides strong evidence for the conclu- sion that similar promoters are employed in the two organisms. Furthermore, the promoter recognition regions (2.4 and 4.2) in the SigE, SigG, and SigK sequences are 100% conserved compared with B. subtilis. The gene encoding the mother cell-specific orK analog of C. acetobutylicum is specified by a single undisrupted open reading frame, as in B. thuringiensis (1). This observation confirms the demonstration that the chromosomal rearrangement of sigK in B. subtilis is not an essential feature for sporulation (37). However, the 5-bp recombination site sequence (5'-AATGA-3') ( 65) is 100% conserved. On the basis of Northern blot analysis, we were unable to demonstrate differential expression of the sequenced cr factor homologous genes, which might be due to cross- hybridization of these conserved genes with the probes used. Furthermore, only a low sporulation efficiency could be achieved in CAMM medium. 
The data presented here provide strong evidence that many of the complex regulation features of sporulation in B. subtilis seem to be conserved in C. acetobutylicum and possibly Clos- tridium spp., despite different induction patterns of sporulation in Bacillus spp. (nutrient starvation) (20) and C. acetobutylicum (excess nutrients required; induction of solvent production is essential) (42). Thus, regulation of sporulation in these two genera seems to differ from the mycelial, gram-positive genus Streptomyces, in which a a factor with significant homology to the motility a factor D of B. subtilis is required for early sporulation (12). 
A number of investigators have previously suggested common features in regulation of solvent formation and sporula- tion in C. acetobutylicum (33, 41-43, 49, 57). These two processes are not inextricably linked, though their mechanisms of induction do have features in common, as indicated by the loss of spore-forming potential and the loss of solvent forma- tion ability in some mutants (33,42,43,57). Furthermore, motility (highest motility is observed during acidogenesis and declines with the onset of solventogenesis) and solventogenesis appear to be linked to some extent (26,27,53). The findings reported here, that hybridization signals were obtained in strain DSM 792 with a probe complementary to the motility a factor D of B. subtilis, indicate the presence of such a af factor in C. acetobutylicum. Currently, only vague speculations are possible about the regulatory network controlling motility, solventogenesis, and sporulation. However, the data presented here suggest that at least sporulation and possibly motility are regulated by alternative ar factors. Solventogenic genes are most likely not regulated by a similar mechanism since their promoters have considerable sequence homology with the o'-dependent promoters (21,23,59,73). Previously reported data indicated that tRNA CG may play a role in the transla- tional control of several inducible C. acetobutylicum metabolic networks (60). However, the nucleotide sequences of the dnaE and sigA genes contain three and one ACG codon, respec- tively. It is highly probable that the dnaE and sigA genes are constitutively expressed in C. acetobutylicum, thus rendering the foregoing translational control hypothesis less likely. Work currently performed in several laboratories is aimed at eluci- dating the molecular events leading to initiation of solvento- genesis and sporulation. 

FIG. 1 .

 1 FIG. 1. Nucleotide sequence of the dnaE-sigA operon region. The deduced amino acid sequences for both genes are shown below the DNA sequence. The putative ribosome-binding (Shine-Dalgarno [SD]) site is boxed, and potential stem-loop structures are marked by arrowheads below the sequence. The start point of the transcript as identified by primer extension analysis is marked by an horizontal arrow. Putative promoter regions are overlined. 


FIG. 2 .

 2 FIG. 2. Time course of cell count and the occurrence of motility, forespores, and spores (A) and pattern of product formation and pH (B) during a batch cultivation of C. acetobutylicum DSM 1731 in CAMM medium. (A) 0, cell count. (B) *, acetate; 0, acetone; El, butanol; *, butyrate; A, ethanol; *, pH. Arrows indicate the time points of cell harvest for subsequent RNA isolation. 




  . 




  The 


FIG. 4 .

 4 FIG.4. Identification of the 5' end of the C. acetobutylicum dnaE- sigA operon by primer extension analysis. Oligonucleotides a4336 (a) and u4342 (b), complementary to the 5' region of dnaE, were annealed to 10 ,ug of total RNA from acidogenic (lanes Ac) and solventogenic (lanes S) continuous culture and 3 ,ug of batch culture (Fig.2) C acetobutylicum DSM 1731 cells. The primer-extended products were coelectrophoresed with a sequencing ladder generated with U4336 as the primer. The arrows on each side indicate the positions of the products obtained with both primers. 


FIG. 5 .

 5 FIG. 5. Schematic representation of the cloned fragment in plasmids pSE1 and pT22. Arrows indicate directions of transcription, positions, and sizes of open reading frames. Numbers representing the nucleotide positions and restriction endonuclease sites are marked below the bar. 




  Consensus ------- --..RRRR. C..C..R.TT E........ 


E

  FIG. 8. Nucleotide sequence of the sigK DNA region. The predicted amino acid sequence for the gene is shown below the DNA sequence. Potential stem-loop structures are marked by arrowheads below the sequence. A putative ribosome-binding (Shine-Dalgarno [SD]) site is boxed. 


TABLE 1 .

 1 Amino acid similarity of DnaE from C. acetobutylicum to corresponding proteins of other bacteriaa Similarity data were derived from pairwise alignments with the complete amino acid sequence of DnaE from C. acetobutylicum.The symbol comparison table ofGribskov and Burgess (25)was used to calculate percentage identity and similarity. 


	Organism	Protein	Identity	Similarity Reference
	Eschenchia coli	DnaG	33	56	9
	Salmonella typhimurium DnaG	31	54	19
	Rickettsia prowazekii	DnaG	29	53	46
	Buchnera aphidicola	DnaG	24	51	38
	Bacillus subtilis	DnaE	30	55	74

a 
TABLE 2 .

 2 Amino acid similarity of SigA from C. acetobutylicum to selected corresponding proteins of other bacteriaa 


	Organism	Protein	Identity Similarity Reference
	Bacillus subtilis	RpoD	69	83	24
	Staphylococcus aureus	PlaC	69	81	5
	Salmonella typhimurium SigA	58	76	19
	Escherichia coli	RpoD	58	76	8
	Streptomyces coelicolor	HrdB	54	68	62
	Chlamydia trachomatis	SigA	53	72	18
	Streptomyces coelicolor	HrdA	48	68	67
		HrdC	47	64	67
		HrdD	45	67	67

(25)milarity data were derived from pairwise alignments with the complete amino acid sequence of SigA from C. acetobutylicum.The symbol comparison table of Gribskov and Burgess(25)was used to calculate percentage identity and similarity. 
TABLE 3 .

 3 Amino acid similarity of SpoTIGA, SigE, SigG, and SigK from C. acetobutylicum to corresponding proteins of bacillia 


			SpoIIGA	SigE		SigG		SigK		
	Organism	Identity	Similarity	Identity	Similarity	Identity	Similarity	Identity	Similarity	Reference(s)
		(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	
	B. subtilis	23	52	72	86	74	88	57	76	48, 63-65
	B. thuringiensis	24	51	69	84	80	88	58	77	1



  (69)E. coli nusB region(69). Identical amino acids in all four proteins are boxed. A consensus sequence is given above the alignment if the respective amino acid occurs in at least three proteins. 


				-
	EC	MHCPFCFAVD TKVIDSRLVG EGSSVRRRRQ CLVCNERFTT FEVAELVMPR
	Ca Tt Bs EC	V.K. E. F...K VIKKDSSREF FDKSKVIfl IKSC KRPWsI RQQIEDIASD VEKSISNQMV E.. A. ..RPV .....E. . VVKRDGRKEP FNPDKLL4 LLACEKRWC EEVLRRFAYT FEDQVSG------------L IKAC R LKTLEDMCFD IEKELRNQGC VVKSNDVREP FNEEKLR. 4 l EKRPĩ SDDVEHAINH IKSQLRATGE
	Ca Tt Bs Ec	A MVMEKLKE M V 5E M]EDRLAICM 1 E V} 3KM] LVMEQ	ASV Y F.DI FAVY 2I ASVF FI	..LNLVNK IRCPRKP II WIKK G}ARLED-
	FIG. 7. Amino acid sequence alignment of the putative open
	reading frame V gene product of C. acetobutylicum (Ca) with putative
	proteins encoded in the following DNA regions: Tt, T. thermophilus
	16S rRNA region (29); Bs, B. subtilis dnaB region (partly sequenced)
	(32);			

ACKNOWLEDGMENTS

We are grateful to reviewer 3 for critical analysis of our original submission as well as many helpful suggestions, which undoubtedly required much time and effort. 
J. D. Santangelo acknowledges financial support obtained from the Alexander-von-Humboldt Stiftung. This work was supported by a grant from the Deutsche Forschungsgemeinschaft.
Uwe Sauer, Anke Treuner, Malte Buchholz, Joseph D Santangelo, Peter Durre
Sporulation and Primary Sigma Factor Homologous Genes in Clostridium acetobutylicum