Food Science Department, Cornell University, Ithaca, NY 14853, USA1
Author for correspondence: Kathryn J. Boor. Tel: +1 607 255 3111. Fax: +1 607 254 4868. e-mail: kjb4{at}cornell.edu
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ABSTRACT |
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Keywords: sigma factors, sporulation, sigma E, Bacillus spp., Clostridium spp
The GenBank accession numbers for the sequences determined in this work are AF225461AF225466.
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INTRODUCTION |
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Transcription of B. subtilis genes required for sporulation is principally regulated by a cascade of sigma factors including the sporulation-specific sigma factors F,
E,
G and
K (Errington, 1993
; Haldenwang, 1995
; Stragier & Losick, 1996
). Sigma factors are a class of proteins constituting essential dissociable subunits of prokaryotic RNA polymerase. They provide promoter recognition specificity, contribute to DNA strand separation, then dissociate from RNA polymerase core enzyme following transcription initiation (Aiyar et al., 1994
; Haldenwang, 1995
; Helmann & Chamberlin, 1988
).
In the predivisional B. subtilis cell, the two-cistron spoIIG operon comprised of spoIIGA and spoIIGB is transcribed by the A holoenzyme (Baldus et al., 1994
; Buckner et al., 1998
).
E, encoded by spoIIGB, is synthesized as an inactive pro-protein (pro-
E) in the predivisional cell (Errington, 1993
). Pro-
E becomes active by cleavage of approximately 27 amino acids from its amino terminus following asymmetric cell division (Carlson et al., 1996
; Peters et al., 1992
). This SpoIIGA-mediated proteolytic processing event is triggered by the extracellular signal protein SpoIIR that is produced in the forespore under the control of
F (Hofmeister et al., 1995
; Karow et al., 1995
; Londono-Vallejo & Stragier, 1995
).
E activity is confined to the mother cell, where it directs transcription of several genes including spoIIID (Errington, 1993
). SpoIIID is a sequence-specific DNA-binding protein that regulates transcription of many genes of the
E and
K regulons (Halberg & Kroos, 1994
; Halberg et al., 1995
; Zhang et al., 1997
).
Several lines of evidence suggest that various regulatory features of the sporulation pathway are conserved between Clostridium spp. and Bacillus spp. Clostridium acetobutylicum ORFs bearing significant identity to the Bacillus genes encoding the sporulation sigma factors E,
G and
K have been identified (Sauer et al., 1994
; Wong et al., 1995
) and expression patterns of these genes have been shown to be similar to those of B. subtilis (Santangelo et al., 1998
). As in B. subtilis, C. acetobutylicum has a two-cistron spoIIG operon comprised of spoIIGA and spoIIGB. In addition, genes bearing identity with B. subtilis spoOA and spoIVB have been identified in six Clostridium species (Brown et al., 1994
).
An improved understanding of sporulation processes in Clostridium spp. will also provide insight into physiological aspects unique to this genus. For example, linkages appear to exist between sporulation and toxin production in Clostridium spp. To illustrate, Zhao & Melville (1998) found that the P2 and P3 promoters of the enterotoxin gene (cpe) of C. perfringens bear similarity to the consensus promoter sequence for Bacillus spp.
E-dependent genes.
This study was designed to further improve our comparative knowledge of the sporulation processes in Bacillus and Clostridium. We used spoIIGB sequences from various spore-formers to investigate evolutionary relationships among these bacteria. We also used E to investigate the functional conservation of the sporulation pathway between C. acetobutylicum and B. subtilis.
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METHODS |
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PCR products were purified using the Wizard PCR preps DNA purification system (Promega) after excision of the band from a low-melting agarose gel (Fisher Scientific). The purified PCR products were cloned into the pCR2.1 vector and transformed into competent E. coli INVF' as specified in the TA Cloning Kit (Invitrogen). Plasmids were purified with the QIAprep spin plasmid purification kit (Qiagen) and at least two clones per bacterial strain were sequenced using the primers M13F and M13R. Sequencing was performed using an Applied Biosystems Division 373 A Stretch DNA sequencer (Perkin Elmer) at the Cornell Center of Advanced Technology.
Sequence alignments and phylogenetic trees.
Alignments and phylogenetic analyses were performed using the programs of the DNAStar software package and the PHYLIP software package, version 3.57c (Felsenstein, 1989 ), respectively.
Bacterial transformation.
Preparation and transformation of competent B. subtilis cells were performed following the two-step transformation procedure (Cutting & Horn, 1990 ). Transformants were selected on TBAB agar (Difco) plates containing 5 µg chloramphenicol ml-1 (for pDH32 and pKSV7) or 5 µg neomycin ml-1 (for pPL703).
Generation of the B. subtilis E null mutant.
A nonpolar internal deletion mutation in spoIIGB of B. subtilis PB2 was generated using splicing by overlap extension (SOEing) PCR (Horton et al., 1990 ) and the vector pKSV7 (Smith & Youngman, 1992
). SOEing PCR primers were designed based on published sequences of spoIIG and spoIIIG of B. subtilis (GenBank accession nos X17344, X01180 and X57547). The primers SOE-A and SOE-B (Table 2
) amplified a 423 bp fragment encoding portions of spoIIGA and spoIIGB. The primers SOE-C and SOE-D amplified a 420 bp fragment encoding portions of spoIIGB and spoIIIG. The two resulting PCR fragments served as template for a subsequent PCR amplification using SOE-A and SOE-D to yield an 843 bp fragment with an in-frame 603 bp deletion in spoIIGB. This PCR fragment was digested with XbaI and EcoRI and cloned into pKSV7. The resulting plasmid, pEFA60, was transformed into competent B. subtilis PB2. Subsequently, a chloramphenicol-resistant transformant was serially passaged in LB with 5 µg chloramphenicol ml-1 at 42 °C to direct chromosomal integration of the plasmid at the B. subtilis spoIIG locus by homologous recombination. Following the third passage, a loopful of culture was streaked onto TBAB with 5 µg chloramphenicol ml-1 and incubated at 42 °C. Colonies were passaged in LB (with no chloramphenicol) at 30 °C. Following every third passage, the culture was screened for plasmid excision by replica plating on TBAB with and without chloramphenicol. Allelic-exchange mutagenesis was confirmed by PCR amplification and by directly sequencing the PCR product.
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Single-copy chromosomal complementation of the B. subtilis E null mutant.
Derivatives of the vector pDH32 were constructed for integration of spoIIGBs or spoIIGCa into the amyE chromosomal locus of the B. subtilis strain FSL-A2-030 (sigE) (Henner, 1990
). DNA fragments containing spoIIG were amplified with primers CAF1 and CAR6 for C. acetobutylicum and with primers BSF1 and BSR9 for B. subtilis PB2 (Table 2
). The purified fragments were cloned into EcoRIBamHI sites of pDH32, generating lacZ transcriptional fusions in the resulting plasmids, pEFA91 and pEFA92. These plasmids were amplified in E. coli DH5
, linearized with PstI and transformed into competent B. subtilis FSL-A2-030 cells with selection for chloramphenicol resistance, generating strains FSL-A2-037 (C. acetobutylicum spoIIG::amyE) and FSL-A2-038 (B. subtilis spoIIG::amyE). Disruption of the amyE locus was determined by flooding TBAB plates containing 1% soluble starch with Lugols iodine: Amy+ colonies were surrounded by a zone of clearing in the medium; Amy- colonies were not. Expression of the resulting spoIIGlacZ fusions was monitored by measuring ß-galactosidase activity (Miller, 1972
).
Sporulation assay.
B. subtilis cells were inoculated into 10 ml Schaeffers 2xSG liquid medium (Leighton & Doi, 1971 ) supplemented with 25 mg tryptophan ml-1, grown to mid-exponential phase with vigorous shaking (300 r.p.m.) at 37 °C, diluted 1:25 into a final volume of 50 ml of fresh 2xSG and returned to the incubator shaker. Samples were removed after 24 h for parallel assessments of viable counts and of chloroform-resistant cells. Chloroform (25 µl) was added to 2·0 ml samples, then the treated cultures were held at room temperature without shaking for an additional 24 h. Serial dilutions of samples taken both before and after chloroform treatment were plated on 2xSG. Viable cell counts before and after chloroform treatment were used to calculate the sporulation efficiency for each strain: sporulation efficiency=(no. of viable cells after chloroform treatment)x100/(no. of viable cells before treatment) (Boor, 1994
). Presence of spores was also verified using phase-contrast microscopy.
Construction of transcriptional lacZ fusions.
Transcriptional lacZ fusions were constructed to investigate the functional activity of C. acetobutylicum E or B. subtilis
E in the extrachromosomally complemented B. subtilis
E null mutant. For this purpose, a 359 bp DNA fragment containing the
E-dependent promoter region of B. subtilis spoIIID was amplified by PCR with primers BSF8 and BSR8 (Table 2
). The purified PCR product was cloned into pDH32, yielding pEFA90 (Table 1
). After confirming the correct insert by sequencing, the plasmid was linearized and transformed into competent B. subtilis PB2 and FSL-A2-030 (
sigE) cells with selection for chloramphenicol resistance, generating strains FSL-A2-033 and FSL-A2-034. These strains were also screened for AmyE phenotype as described above. Strain FSL-A2-034 was then transformed with either pEFA10 (spoIIGCa) or pEFA11 (spoIIGBs) to generate strains FSL-A2-035 and FSL-A2-036. All resulting strains were assayed for ß-galactosidase activity (Miller, 1972
).
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RESULTS |
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Extrachromosomal complementation of the B. subtilis E null mutant
To assess E function, we determined and compared sporulation efficiencies for the B. subtilis strains FSL-A2-030 (
sigE), FSL-A2-031 (pEFA10
FSL-A2-030), FSL-A2-032 (pEFA11
FSL-A2-030) and the wild-type PB2 (Fig. 5
). The sporulation efficiencies for the strains bearing pEFA10 (spoIIGCa) and pEFA11 (spoIIGBs) (FSL-A2-031 and FSL-A2-032) were ~0·2% for each complemented strain. Restoration of spore production in these strains was verified by phase-contrast microscopy. Phase-bright spores were present in both strain FSL-A2-031 and strain FSL-A2-032 following growth on 2xSG plates for 10 d.
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Chromosomal complementation of the B. subtilis E null mutant
spoIIGBs and spoIIGCa were integrated into the B. subtilis amyE locus utilizing pDH32-derived plasmids pEFA91 and pEFA92, respectively, to allow single-copy complementation of the spoIIGB null mutation and to generate spoIIGBlacZ fusions. Expression of these fusions in strains FSL-A2-037 and FSL-A2-038 was monitored through ß-galactosidase activity measurement, as shown in Fig. 6. Both strains showed detectable ß-galactosidase activity in mid-exponential phase. ß-Galactosidase activity in FSL-A2-038 (amyE:: spoIIGBs) peaked at 3 h after entry into stationary phase. FSL-A2-037 (amyE::spoIIGCa) produced 1885% of the ß-galactosidase activity of FSL-A2-038 between 3 and 8 h after entry into stationary phase, with a peak of activity at 15 h after entry into stationary phase.
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DISCUSSION |
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Sequence analyses
E is a member of the
70 family of proteins. Each of these
proteins is comprised of four regions, which are believed to have distinctive functions and which are conserved to varying degrees among all family members (reviewed by Lonetto et al., 1992
). Alignment of the three complete pro-
E sequences from B. subtilis, B. thuringiensis and C. acetobutylicum obtained from GenBank showed amino acid identities ranging from 90% for region 4.2 to 63·0% for region 1.2 (data not shown). Notably, while the N-terminal amino acid sequence which is cleaved from the pro-
E protein to create the active
E protein (27 amino acids in each Bacillus species; 23 amino acids in C. acetobutylicum) (Carlson et al., 1996
; Peters et al., 1992
) is poorly conserved among these three organisms (21·4 %), the amino acid sequence flanking the cleavage site (YYIGG, amino acids 2731 in B. subtilis) is exactly conserved among all three species.
The partial E sequences obtained in this work as well as the
E sequences from B. subtilis, B. thuringiensis and C. acetobutylicum obtained from GenBank were combined for further sequence and phylogenetic analyses. The multiple alignment of the partial
E sequences (Fig. 1
) includes the C-terminal portion of subregion 2.3 to the beginning of subregion 4.2 of the
70 family of proteins (Lonetto et al., 1992
). Subregion 2.3 is proposed to be involved in catalysing DNA melting during open promoter complex formation (Helmann & Chamberlin, 1988
). Jones & Moran (1992)
demonstrated that a substitution of cysteine to arginine at position 117 in subregion 2.3 of B. subtilis
E conferred many of the characteristics expected of a sigma factor defective in DNA strand opening. This cysteine at position 117 is conserved among all of the
E sequences from the Bacillus, Clostridium and Paenibacillus species examined in this study.
Subregion 2.4, which is involved in RNA polymerase holoenzyme promoter utilization specificity, recognizes the -10 region of the promoter sequence (Lonetto et al., 1992 ). For B. subtilis
E, the methionine at position 124 was shown to interact with the nucleotide at position -13 in the -10 region of the spoIID, spoIIID and cotEP1 promoters (Diederich et al., 1992
; Jones & Moran, 1992
; Tatti et al., 1991
). Also, the glutamic acid residue at position 119 and the asparagine at position 120 appear to interact with nucleotides in the -10 region of
E-dependent promoters (Tatti & Moran, 1995
). The corresponding methionine, glutamic acid and asparagine residues are conserved among all the species that we analysed. Our findings provide further evidence of evolutionary conservation of sporulation mechanisms among members of the diverse genera Bacillus, Paenibacillus and Clostridium (Sauer et al., 1994
; Wong et al., 1995
).
Phylogenetic analysis
The phylogenetic tree (Fig. 2) generated by parsimony analysis of the partial
E amino acid sequences grouped Bacillus spp. and Clostridium spp. into distinct clusters separate from P. polymyxa and C. bifermentans. Collins et al. (1994)
had previously classified C. bifermentans into a cluster (cluster XI) distinct from C. butyricum, C. acetobutylicum, C. perfringens and C. sporogenes (cluster I) based on 16S rRNA analyses. These authors observed that C. bifermentans formed a distinct subgroup along with several other Clostridium spp. within cluster XI, suggesting that this subgroup may represent a different genus. These findings are consistent with our
E analysis, which confirmed a unique phylogenetic position for C. bifermentans. Our data also confirmed the status of P. polymyxa as a representative of a genus separate from Bacillus.
To verify the E phylogenetic analysis, 16S rRNA GenBank sequences for the corresponding species were also analysed by the parsimony method. The resulting phylogenetic tree (Fig. 3
) revealed clustering patterns very similar to those seen with
E. With the exception of C. acetobutylicum and C. sporogenes, multiple sequences from a given species generally clustered closely. The five C. acetobutylicum 16S rRNA sequences formed two different clusters, with three sequences clustering with C. sporogenes and two sequences clustering with C. butyricum. These findings are consistent with a previous phylogenetic analysis that reported distinctively different spoOA sequences from two C. acetobutylicum strains, with one sequence clustering with those of C. butyricum (Brown et al., 1994
). Our findings provide further evidence in support of the reclassification of strains currently recognized as C. acetobutylicum into two phylogenetically distinct lineages and, perhaps, as two different species (Keis et al., 1995
; Wilkinson et al., 1995
). Two of the C. sporogenes 16S rRNA sequences clustered together, while a third C. sporogenes sequence (GenBank accession no. L09175) clustered most closely with the Bacillus cluster. These findings may suggest the existence of two phylogenetically distinct lineages of C. sporogenes.
The data presented here, as well as previous studies that identified homologous genes encoding sporulation-specific proteins in Clostridium spp. and in Bacillus spp. (e.g. spoOA; Brown et al., 1994 ) suggest a common evolutionary ancestor of the sporulation pathways in these two distinctive genera. As an alternative hypothesis, the sporulation pathways and the underlying genes could have evolved independently in these two genera. To further probe these two hypotheses, we performed a phylogenetic analysis of all sigma factor sequences reported for the genera Bacillus, Paenibacillus and Clostridium, including the partial
E sequences described here. The resulting tree constructed by the parsimony method is shown in Fig. 4
. Homologues of sporulation-specific sigma factors (
E,
K and
G) from Bacillus and Clostridium spp. always formed coherent clusters distinct from other sigma factors. These findings suggest a common origin of sporulation sigma factors in the endospore-forming Bacillus and Clostridria rather than independent evolution of these proteins in these genera.
Functional analysis of E conservation
To further probe the evolutionary conservation between the sporulation pathways, specifically E, in Clostridium and Bacillus spp., we performed complementation analyses with the spoIIG alleles from B. subtilis and C. acetobutylicum.
Extrachromosomal complementation.
Derivatives of the low-copy-number plasmid pPL703 were used for extrachromosomal complementation of the B. subtilis E null mutant. Both complemented strains, FSL-A2-031 (spoIIGCa) and FSL-A2-032 (spoIIGBs), showed only partial recovery of the capacity to form spores in 1-d-old cultures (approx. 0·2% sporulation efficiencies).
E activity was assessed by measuring expression from the
E-dependent spoIIID promoterlacZ fusion. The wild-type strain FSL-A2-033 (PB2 bearing a spoIIIDlacZ fusion) yielded ß-galactosidase activity within 2 h after the onset of stationary phase growth, consistent with previous findings (Tatti et al., 1991
). Strains FSL-A2-035 and FSl-A2-036, which bore the spoIIIDlacZ fusion along with spoIIG from either C. acetobutylicum or B. subtilis, respectively, yielded ß-galactosidase activities at levels only slightly higher than that produced by the negative control strain, PB2. These results support previous reports of the ability of low levels of spoIIG activity (~17% of normal) to permit sufficient formation of endospores to allow spore detection under phase-contrast microscopy and production of viable colonies following chloroform treatment of 1-d-old cultures (Schyns et al., 1997
). The absence of full recovery of the wild-type phenotype by the two complemented strains, and particularly by the spoIIGBs-complemented strain, is likely a consequence of our extrachromosomal complementation strategy. Adams et al. (1991)
hypothesized that incomplete complementation of a B. subtilis
K defect with a pPL703 derivative bearing B. thuringiensis
29 resulted from physiologically inappropriate gene copy numbers. In our study, it is possible that the presence of multiple spoIIG copies disrupted optimal levels of gene expression required for spore formation.
Chromosomal complementation.
To probe the influence of spoIIG copy number on complementation of the E null mutation, we performed additional complementation studies using pDH32 derivatives to integrate a single copy of either the B. subtilis or the C. acetobutylicum spoIIG operon into the chromosomal B. subtilis amyE locus of the
E null mutant, FSL-A2-030. Construction of pDH32 derivatives created spoIIGlacZ fusions that allowed us to monitor spoIIG expression in the B. subtilis background. Measurement of ß-galactosidase activity from these fusions showed that expression of spoIIGCalacZ was delayed in comparison with that of spoIIGBslacZ, peaking at 15 h rather than 3 h after entry into stationary phase (Fig. 6
). Colony recovery following chloroform treatment revealed that B. subtilis spoIIG restored the wild-type phenotype, while C. acetobutylicum spoIIG yielded only a low level of complementation (0·0001% sporulation efficiency) in 1-d-old cultures.
Differences in spoIIGBs and spoIIGCa promoter regions may be at least partially responsible for the observed differences in transcription patterns directed by these constructs in the B. subtilis genetic background. In B. subtilis, the spoIIG operon has a A-dependent promoter (Kenney et al., 1988
) that is activated by SpoOA~P during the onset of sporulation (Baldus et al., 1994
; Buckner et al., 1998
). The promoter region of the C. acetobutylicum spoIIG operon was recently mapped by primer extension analysis (Santangelo, 1998
). The deduced -35 and -10
A recognition sequences in this operon differ from those in B. subtilis by 2 and 1 nucleotides, respectively, and the spacing between them consists of 21 nucleotides instead of the 23 present in the B. subtilis spoIIG. Further, two SpoOA-binding sites centred at -87 and -35 exist upstream of B. subtilis spoIIG (Baldus et al., 1994
; Satola et al., 1991
, 1992
) but only one possible site was deduced in the C. acetobutylicum homologue (Wong et al., 1995
).
Transcription and activation of E is very complex (Stragier & Losick, 1996
). Our findings suggest that regulation of spoIIG expression may differ between Clostridium spp. and Bacillus spp., as spoIIGCa is expressed later than spoIIGBs in the B. subtilis genetic background. It is also possible that spoIIGCa does not fully complement the B. subtilis
E null mutation as a consequence of inefficient association of C. acetobutylicum
E with the B. subtilis RNA polymerase or due to an inability of C. acetobutylicum
EB. subtilis RNA polymerase holoenzyme to efficiently transcribe from
E-dependent promoters in B. subtilis. Genetic analyses of C. acetobutylicumB. subtilis hybrid spoIIG sequences will provide insight into these hypotheses.
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ACKNOWLEDGEMENTS |
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The authors gratefully acknowledge the technical expertise of Brian Miller, Micaela Chadwick and Michael Gray.
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Received 19 August 1999;
revised 25 January 2000;
accepted 3 April 2000.