Department of Microbiology, University of Otago, Dunedin, New Zealand1
Author for correspondence: David T. Jones. Tel: +64 3 479 7735. Fax: +64 3 479 8540. e-mail: david.jones{at}stonebow.otago.ac.nz
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ABSTRACT |
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Keywords: solvent-producing clostridia, PFGE, 2D-PFGE, linking library, genome organization
Abbreviations: 2D, two-dimensional
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INTRODUCTION |
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The first successful industrial solvent-producing strains to be isolated, patented and used for the large-scale production of solvents from starch-based substrates were classified as C. acetobutylicum. Following the switch to molasses as the preferred fermentation substrate for commercial fermentation during the mid-1930s, numerous new saccharolytic, solvent-producing clostridial strains were isolated that performed more efficiently on these sugar-based substrates. Many of these new industrial strains were patented under novel species names but none of these were recognized as legitimate species. Once the acetone-butanol fermentation process declined during the latter part of the twentieth century these names fell into disuse. Subsequently, the majority of the industrial solvent-producing clostridial strains became designated as either C. acetobutylicum or Clostridium beijerinckii. Recent studies have, however, revealed that the various strains of industrial solvent-producing clostridia belong to four distinct species: C. acetobutylicum, C. beijerinckii, C. saccharobutylicum and Clostridium saccharoperbutylacetonicum (Johnson et al., 1997 ; Keis et al., 1995
), with the latter two species in the process of being formally classified (S. Keis, R. Shaheen & D. T. Jones, unpublished results).
Most of the studies undertaken over the last two decades have focused on strains belonging to the species C. acetobutylicum and C. beijerinckii, which are phylogenetically only distantly related (Johnson et al., 1997 ; Keis et al., 1995
). Physical and genetic maps of the chromosomes of these two species have been published (Cornillot et al., 1997
; Wilkinson & Young, 1995
), and the entire C. acetobutylicum genome sequence has now been determined (http://www.genomecorp.com/sequence_centre/bacterial_genomes/).
Industrial strains belonging to the recently named C. saccharobutylicum species were amongst the most successful saccharolytic, solvent-producing clostridia utilized for the commercial production of solvents from molasses (Jones & Keis, 1995 ; Keis et al., 1995
; Shaheen et al., 2000
). Nevertheless, C. saccharobutylicum has been less intensively studied than the other two solvent-producing species and currently little is known about the genomic structure and organization of this organism.
In this study, we have used one- and two-dimensional (2D) PFGE, BssHII linking clones, and Southern hybridization analysis to construct a physical and genetic map of the C. saccharobutylicum type strain, NCP 262. The size of the circular chromosome was estimated to be 5·3 Mb and it contains 12 rrn operons that are transcribed divergently from the origin of replication. The genetic map of this species was compared with those of the spore-forming bacteria Bacillus subtilis, C. acetobutylicum, C. beijerinckii and Clostridium perfringens.
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METHODS |
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For 2D digestions, a 4-mm-wide slice of the lane, containing the first digestion, was equilibrated in a 15 ml polypropylene tube for 30 min at 4 °C with 2 ml of the second restriction enzyme buffer. The DNA slice was then preincubated overnight at 4 °C in 2 ml restriction enzyme buffer containing 100 U enzyme to allow the enzyme to diffuse into the agarose slice. This was followed by incubation in a Hybaid oven at the appropriate temperature with slow rotation for 3 h.
Pulsed-field and agarose gel electrophoresis.
PFGE was performed in a contour-clamped homogeneous electric field (CHEF) electrophoresis system (CHEF-DRIII, Bio-Rad). Agarose plugs were equilibrated three times in 0·5x TBE buffer (45 mM Tris/borate, 1 mM EDTA) for 15 min prior to loading agarose (Molecular Biology Grade; Bio-Rad) gels. Gels were routinely subjected to electrophoresis at 6 V cm-1, 14 °C, at an included angle of 120°. Agarose concentrations, pulse times and total running times varied according to the size range of fragments to be separated; gel electrophoresis conditions for individual experiments are described in the figure legends. Lambda DNA concatemers with lambda HindIII-digested fragments (Low Range PFG Marker; New England Biolabs), lambda concatemers (Lambda Ladder PFG Marker; New England Biolabs), and chromosomes of Saccharomyces cerevisiae YNN295 (Bio-Rad) were used as molecular size standards. Conventional agarose gels were subjected to electrophoresis at 1·4 V cm-1 for 18 h and lambda DNA digested with HindIII was used as a size standard.
Southern hybridization.
DNA fragments separated by PFGE were transferred onto Hybond-N+ nylon membranes (Amersham) using a VacuGene (Pharmacia-LKB) vacuum blotting system. The DNA was depurinated with 0·25 M HCl for 20 min, denatured (in 1·5 M NaCl, 0·5 M NaOH) for 1 h, and transferred in 20x SSC (1x SSC is 0·15 M NaCl plus 0·015 M sodium citrate) for 2 h. DNA in conventional agarose gels was depurinated, denatured and transferred for 10 min, 10 min and 1 h, respectively.
Plasmid DNA used for probing was isolated with the High Pure plasmid isolation kit (Roche Molecular Biochemicals). PCR products used as probes were extracted from 1% (w/v) SeaPlaque low-melting-point agarose (FMC BioProducts) gels using the QIAEXII gel extraction kit (Qiagen).
DNA was labelled with [-32P]dCTP (Amersham) by random priming using the RTS RadPrime DNA labelling kit (GibcoBRL). Prehybridization and hybridization were carried out in the same hybridization buffer (Church & Gilbert, 1984
) at 65 °C for 18 to 24 h. After hybridization, membranes were washed sequentially for 5 min in 2x SSC/0·1% SDS (37 °C), 20 min in 1x SSC/0·1% SDS (65 °C), and 20 min in 0·1x SSC/0·1% SDS (65 °C) before exposure to X-ray film (Cronex, DuPont). For heterologous probes, membranes were washed less stringently by carrying out the initial wash as above, followed by 20 min in 2x SSC/0·1% SDS at 50 °C.
Genomic library construction and cloning of small BssHII fragments.
Genomic DNA was extracted from agarose plugs using the QIAEXII gel extraction kit (Qiagen). The DNA was sheared by nebulization and end-repaired with T4 DNA polymerase (Roche Molecular Biochemicals) and Klenow (Roche Molecular Biochemicals) (Andersson et al., 1996 ). The end-repaired DNA was separated on a 1% (w/v) SeaPlaque low-melting-point agarose (FMC BioProducts) gel. DNA ranging from 4 to 23 kb was excised from the gel, and the DNA was extracted using the QIAEXII gel extraction kit, followed by ligation into dephosphorylated, SmaI-digested pUC8. For the cloning of small (<5 kb) BssHII fragments, DNA was extracted from BssHII-digested genomic DNA plugs and ligated into dephosphorylated BssHII-digested pNEB193. Electrocompetent E. coli cells were transformed by electroporation and white ampicillin-resistant transformants were selected on media containing X-Gal, IPTG and ampicillin.
Preparation of linking clones.
The kanamycin cassette from pUC4K was amplified using the PCR conditions described below and the primers KANFWD (5'-TCGACTACGCGTAGCTTCACGCTGCCGCAAGC-3') and KANREV (5'-AGTACGACGCGTGGGGTGGGCGAAGAACTCCA-3'), each containing MluI recognition sites (shown in bold) near their 5' ends. This cassette was digested with MluI to create a kanamycin cassette with BssHII-compatible ends.
Plasmid DNA was isolated from the library of pooled recombinant NCP 262 random pUC8 clones using a Qiagen maxi-prep kit. The DNA was digested with BssHII, treated with alkaline phosphatase (Roche Molecular Biochemicals) and ligated with the MluI-digested kanamycin cassette. Clones with C. saccharobutylicum NCP 262 DNA containing the rare BssHII restriction enzyme site were obtained by selection on media containing ampicillin and kanamycin. These linking clones were radiolabelled and hybridized to Southern blots of PFGE-separated restriction digests as described above.
Sequence analysis of linking clones.
Plasmid DNA from linking clones was prepared for sequencing using a Quantum Prep plasmid miniprep kit (Bio-Rad). BssHII linking clones were sequenced with the following specifically designed primers, SEQKANLEFT (5'-CCAGTAGCTGACATTCATCCG-3') and SEQKANRIGHT (5'-AGGTTGGGCTTCGGAATCGT-3'), which were complementary to the DNA sequences at either end of the kanamycin cassette. Sequencing was carried out using a BigDye terminator cycle sequencing ready reaction sequencing kit (Applied Biosystems) and a model ABI377 automated DNA sequencer (Applied Biosystems).
Homology searches of non-redundant databases at the National Centre for Biotechnology Information, using BLASTX and BLASTN (Altschul et al., 1997 ), were done through the WWW BLAST Server (www.ncbi.nlm.nih.gov).
Confirmation of BssHII-linked fragments by PCR.
Synthetic primers, based on sequences obtained from linking clones, were designed to amplify products of approximately 400 bp which spanned NCP 262 genomic BssHII sites. PCR was carried out using the Expand High Fidelity PCR system (Roche Molecular Biochemicals) and NCP 262 genomic DNA embedded in agarose plugs was used as the template. The PCR programme consisted of an initial cycle of denaturation (94 °C for 2 min), annealing (50 °C for 2 min) and extension (72 °C for 5 min). This was followed by 30 cycles of 94 °C for 30 s, 50 °C for 30 s and 72 °C for 1 min, and a final cycle of 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 5 min. PCR products were purified, radiolabelled and hybridized to PFGE-separated restriction digests as described above. Purified PCR products were also sequenced as described above with their respective PCR primers.
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RESULTS |
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The enzyme ApaI was also found to yield a suitable number of fragments (1220), several of which co-migrated with those of I-CeuI. This is due to the presence of an ApaI site in the rrs gene of the rrn operon, as confirmed by analysis of the 16S rRNA gene sequence of NCP 262 (GenBank accession number U16147; Keis et al., 1995 ). Since ApaI sites would lie in close proximity to I-CeuI sites in most instances, ApaI was not used for mapping.
The size of the C. saccharobutylicum genome was estimated to be 5·3 Mb by summation of the fragments obtained from individual BssHII, I-CeuI, Sse8387I and RsrII digestions (Table 2) and from BssHII/I-CeuI, I-CeuI/Sse8387I and BssHII/Sse8387I double digestions (Table 3
, Fig. 1
). The fragment sizes were determined from at least five different gels using different PFGE parameters. Attempts to resolve the CeuA and RsrA fragments using PFGE conditions recommended by Bio-Rad for the separation of Hansenula wingei and Schizosaccharomyces pombe chromosomes were not successful. Hence, the sizes of CeuA and RsrA were obtained by analysis of double digests. Fragments SseA and SseB could not be separated (Fig. 1c
), and as a consequence their sizes were also determined more accurately from double digestions. The smallest BssHII fragment (BssO) was not observed in pulsed-field gels nor in conventional agarose gels but was detected by hybridization (see Fig. 3
).
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This indirect end-labelling approach was then combined with 2D-PFGE. The NCP 262 genome was digested with I-CeuI and separated by PFGE in the first dimension, followed by digestion with BssHII and separation in the second dimension (or vice versa) (Fig. 2a). This allowed the arrangement of the I-CeuI restriction sites to be determined by identifying the BssHII fragments that overlapped each I-CeuI site. CeuA, CeuB, CeuC, CeuD and CeuE were the only I-CeuI fragments cleaved by BssHII (Table 4
). Inversely, BssB, BssC, BssF, BssH and BssJ were the only fragments cut by I-CeuI (Fig. 2
). BssB was cut by I-CeuI yielding two fragments of 577 kb (BC1) and 53 kb (BC7), which hybridized with the 3' rrl and rrs probe, respectively (Fig. 2b
). The 577 kb fragment was also found on a 2D pulsed-field gel when CeuC was cleaved by BssHII in the second digestion, whereas the 53 kb fragment was obtained after BssHII digestion of CeuD (Table 4
). Therefore, BssB spans the I-CeuI site that joins CeuC and CeuD. CeuD produced a second fragment of 262 kb (BC3) after BssHII digestion (Table 4
) and this fragment, which hybridized with the 3' rrl probe, was also released from BssF after I-CeuI digestion (Fig. 2b
). BssF also yielded a rrs-specific 114 kb (BC6) fragment after I-CeuI digestion (Fig. 2b
) and this fragment was also obtained after BssHII digestion of CeuE (Table 4
); hence, BssF overlaps CeuD and CeuE. CeuE produced a 3' rrl-specific 53 kb (BC8) end-fragment when digested with BssHII (Table 4
) and this fragment was also found when BssH was digested by I-CeuI and separated in the second dimension (Fig. 2b
). Besides this 53 kb end-fragment, the BssH fragment not only contained the seven I-CeuI fragments CeuF to CeuL, it produced another end-fragment of 26 kb (BC10) after I-CeuI digestion which also hybridized only with the 3' rrl probe (Fig. 2b
). This indicated that the rrn operons contained within this BssH fragment are confluently transcribed from CeuG, which hybridized only with the rrs probe and is contained within BssH. The order of the I-CeuI fragments within BssH was not resolved.
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Alignment of the Sse8387I fragments
The construction of the I-CeuI backbone of the genome of C. saccharobutylicum NCP 262 and the placement of a number of the BssHII fragments enabled the Sse8387I fragments to be located on the physical map. Double digestions of I-CeuI and Sse8387I generated six fragments (CS1 to CS6) (Table 3). SseA, SseB and SseE were cleaved by I-CeuI, and CeuA, CeuC and CeuD were cleaved by Sse8387I (Fig. 1a
, c
). Since CeuC and CeuD were linked and both were cleaved by Sse8387I, a Sse8387I fragment had to overlap this CeuC-CeuD linkage. The only Sse8387I fragment of the appropriate size that was cleaved by I-CeuI was SseE (362 kb), which produced the fragments CS4 (186 kb) and CS5 (176 kb). Subtracting the 186 kb fragment from CeuD (316 kb) left a 130 kb fragment and this accounted for the new CS6 fragment. Hence, it could be concluded that the CS5 fragment was situated within CeuC (675 kb). The remaining size difference of 499 kb for CeuC did not account for any of the new fragment sizes that resulted from a I-CeuI/Sse8387I double digestion. Partial Sse8387I digestions produced fragments of about 395 kb and 430 kb (data not shown), and the only combination of linked Sse8387I fragments that would result in these sizes would be SseE-SseG and SseE-SseG-SseG', respectively. Therefore, SseG and SseG' were located within CeuC, and the new 430 kb (CS2) fragment accounted for the remaining size difference. Since CeuC was linked to CeuB, and the latter was not cleaved by Sse8387I, SseA had to bridge CeuB to produce a new fragment (CS3) of around 300 kb. Similarly, the eight I-CeuI fragments CeuE to CeuL did not contain Sse8387I sites, and the only Sse8387I fragment cleaved by I-CeuI large enough to produce the remaining new 1100 kb fragment (CS1) was SseB. SseF, SseC and SseD were not cleaved by I-CeuI and were therefore contained within CeuA. SseF (194 kb) was cleaved by BssHII producing fragments of about 168 kb (SB6) and 31 kb (SB10), which placed it adjacent to SseA. The orientation of SseC and SseD could not be resolved until the position of the RsrB fragment had been determined.
Alignment of RsrII fragments
Double digestions of the NCP 262 genome with RsrII and I-CeuI produced two new fragments of 1619 kb (RC1) and 726 kb (RC2), with all of the resolvable I-CeuI fragments remaining intact as well as the RsrB fragment (Table 3). Therefore, RsrB had to be located within CeuA. RsrB was placed more accurately on the physical map by analysis of a RsrII/Sse8387I double digestion. This digest generated four new fragments (Table 3
), with SseB, SseD and RsrB being cleaved. RsrB (563 kb) was cleaved by Sse8387I to produce the fragments RS2 (330 kb) and RS4 (224 kb), with SseD overlapping the RsrB fragment by 224 kb and producing the third 260 kb fragment (RS3). The fourth 1252 kb fragment (RS1) was the result of SseB containing the second RsrII site.
Positioning of the single SfiI site on the physical map
The unique SfiI site was located on the physical map from the analysis of a BssHII/SfiI double digestion, which resulted in BssJ (219 kb) being cleaved into two new fragments of about 195 kb and 25 kb (data not shown). The SfiI site was placed more accurately by the analysis of a SfiI/I-CeuI double digestion, which generated two fragments of approximately 160 kb and 880 kb, resulting from the cleavage of CeuB (data not shown).
Ordering of the BssHII fragments on the physical map
The construction of the I-CeuI backbone of the NCP 262 genome resulted in the positioning of five BssHII fragments which were cleaved by I-CeuI, namely BssC, BssJ, BssB, BssF and BssH, with the latter three being linked (see Fig. 5). Another strategy was employed to determine the order and position of the remaining BssHII fragments. This involved the preparation of a library of BssHII linking clones. To construct linking clones, pooled recombinant NCP 262 random pUC8 clones were digested with BssHII and ligated to a kanamycin cassette which had BssHII-compatible ends as described in Methods. These BssHII linking clones were used as probes to determine which BssHII fragments they hybridized with. Sequence was obtained from NCP 262 DNA flanking the kanamycin cassette in linking clones, and was used to carry out BLASTX and BLASTN searches. Linkages were confirmed when sequences flanking a BssHII site showed similarity to the same gene or protein, indicating that it spanned the site. Five linkages were established using this strategy (Table 5
), with linking clone A10 reconfirming the linkage of BssF to BssH as already determined during the construction of the I-CeuI backbone. Sequences obtained from linking clones 26, 27, B10, B12, C4 and D4 (Table 5
) showed no significant similarity to genes or proteins in the genebank databases. Therefore, linkages obtained using each of these clones were confirmed by PCR and Southern hybridization. Primers were designed from sequences obtained from each linking clone to amplify approximately 400 bp regions spanning BssHII sites from genomic DNA. The resulting PCR products were then used as probes to confirm linkages of the BssHII fragments.
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In a number of cases, linking clones hybridized to more than two BssHII fragments due to the presence of repetitive DNA in the genome. Probes derived from short PCR products which spanned BssHII sites were therefore used to determine actual linkages. For example, linking clone B10 hybridized with BssD, BssH and BssM, whereas a PCR product obtained from genomic template DNA using primers designed from the sequence flanking the BssHII site of B10 hybridized only with BssD and BssM. However, linking clone B7 hybridized with fragments BssB, BssF, BssG, BssJ and BssN, and a 378 bp PCR product spanning the BssHII site in linking clone B7 hybridized with BssB, BssG and BssN, with the latter fragment appearing at higher intensity (Fig. 4a). A BLAST search of the sequences flanking the BssHII site from linking clone B7 revealed strong similarity to the IS element ISCb1 from C. beijerinckii (Table 5
). Analysis of the ISCb1 sequence showed that it contained a BssHII site (Liyanage et al., 2000
). We found that clone pEK30, containing a region of ISCb1 spanning the BssHII site, hybridized with BssB, BssG and BssN, with BssN again hybridizing at higher intensity (data not shown). To determine the copy number of the ISCb1-like IS element found on NCP 262, the PCR product (378 bp) obtained from B7 was used to probe genomic digestion products obtained using the frequent-cutting enzymes EcoRV, HindIII and XbaI. The recognition sites for these enzymes were not present in the PCR product. Two fragments from each digest hybridized to the probe, indicating that two copies of the IS element are present on the NCP 262 genome (Fig. 4b
). Hence, it was concluded that one copy of the IS element linked BssN to BssB and a second copy linked BssG to a second fragment, the same size as BssN, designated BssN' (Fig. 5
).
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Wilkinson & Young (1995) reported that some macrorestriction fragments in electrophoretograms from C. beijerinckii were overrepresented when compared with fragments of similar size. They suggested that fragments flanking the origin of replication are overrepresented due to the presence of multiple replication forks in genomic DNA extracted from cells during the exponential growth phase. The genomic DNA from NCP 262 was also isolated from cells in the exponential growth phase and the fragments BssB, BssD, BssF and BssH, along with SseE, were found to be overrepresented in electrophoretograms, whereas the fragments BssC and SseF were underrepresented (Fig. 1
). This suggested that the former fragments were near the origin of replication, whereas the latter two fragments were close to the replication terminus, and this was confirmed by the mapping data presented here.
Location of genetic markers on the chromosome
Cloned genes of homologous and heterologous origin (Table 1) were used to probe Southern blots of single and double digestions to determine gene positions on the physical map of the C. saccharobutylicum NCP 262 genome, and further confirmed the order, position and relatedness of macrorestriction fragments (Fig. 5
). Genes identified from the linking clones (Table 5
) do not appear on the map. Their positions are precisely known, in contrast to those shown, whose approximate positions have been determined by hybridization. The order of genes and/or operons assigned to the same fragment are arbitrary.
The positions of house-keeping genes, and genes involved in the heat-shock response and sporulation were determined, as was the location of some of the genes involved in electron transport and solvent production (Fig. 5). The C. acetobutylicum butanol dehydrogenase isozyme genes (bdhAB) and coenzyme A (CoA)-transferase genes (ctfAB) did not hybridize to the genome of NCP 262.
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DISCUSSION |
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The map was assembled using a number of complementary methods including PFGE, analysis of single and double digestion products, along with Southern hybridization, PCR and DNA sequence analysis of linking clones. The I-CeuI backbone was assembled using rrs- and 3' rrl-specific probes located on either side of the I-CeuI site and reciprocal separation of BssHII and I-CeuI digests by 2D-PFGE. C. saccharobutylicum NCP 262 has 12 rrn operons located on 46% of the chromosome transcribed divergently from CeuG. At present, this is the second-highest number of rrn operons reported, with C. beijerinckii NCIMB 8052 containing 14, the most reported to date for a eubacterium (Wilkinson & Young, 1998 ). Multiple copies of rrn operons are often found in bacterial species capable of rapid growth (Krawiec & Riley, 1990
). C. saccharobutylicum has a mean generation time of about 30 min under optimal conditions, a characteristic which favoured its use in industrial solvent production (McCutchan & Hickey, 1954
).
The B subunit of DNA gyrase (gyrB) is located near the origin of replication in bacterial chromosomes (Salazar et al., 1996 ), and the C. acetobutylicum gyrB probe (Table 1
) hybridized to CeuG, which also hybridized only with the rrs-specific probe and not the 3' rrl-specific probe. Hence, the 12 rrn operons of C. saccharobutylicum are transcribed confluently with the direction of chromosomal replication, which proceeds bidirectionally from an origin of replication located within CeuG to a terminus located within CeuA, and multiple copies of rrn operons are clustered close to the replication origin.
The juxtaposition of the majority of the BssHII fragments was determined using a library of BssHII linking clones and Southern hybridization. Linkages were also confirmed by analysis of sequence obtained from regions flanking the BssHII sites present in linking clones. This analysis also allowed the mapping of a number of genes not previously reported for NCP 262 as listed in Table 5. Many of the linking clones detected more than two BssHII fragments when used as probes, indicating the presence of repetitive DNA on the NCP 262 chromosome. Analysis of one such linking clone led to the discovery of two copies of an insertion sequence very similar to ISCb1, an insertion sequence of the IS4 family isolated from C. beijerinckii NCIMB 8052 which contains a BssHII site (Liyanage et al., 2000
).
The genes involved in controlling electron flow play an important role during the complex biphasic fermentation of carbohydrates to solvents in solvent-producing clostridia. Two of these genes, the iron-only hydrogenase (hydA) and the metronidazole susceptibility (sum) gene, were found to be near the replication origin in C. saccharobutylicum, with the former gene also reported to be close to the origin of replication in C. acetobutylicum (Cornillot et al., 1997 ).
Some of the genes involved in the acid- and solvent-forming pathway were found to be clustered in a small region, representing only 2·5% of the NCP 262 genome. This included the butyryl-CoA synthesis (BCS) operon genes, crt, bcd, etfAB and hbd, which reduce acetoacetyl-CoA to butyryl-CoA in three consecutive reactions, and the butyrate operon genes, ptb and butK, involved in the conversion of butyryl-CoA to butyrate. The BCS operon was found to be in close proximity to the groESL heat-shock operon, as is the case for both C. acetobutylicum (Cornillot et al., 1997 ) and C. beijerinckii (Wilkinson & Young, 1995
). The adh1 gene isolated from C. saccharobutylicum NCP 262 was reported to be downstream from the hbd gene (Youngleson et al., 1989
); hence it was located on the same region as the BCS operon. This NADPH-dependent alcohol dehydrogenase was shown to have activity using butanol and ethanol as substrates (Youngleson et al., 1989
). The genes involved in the final steps of solvent production are the aldehyde/alcohol dehydrogenase (aad) and CoA-transferase (ctfAB) genes, which form the sol operon (Dürre et al., 1995
), along with the acetoacetate decarboxylase (adc) and butanol dehydrogenase (bdhAB) genes. Toth et al. (1999)
reported that a probe derived from the C. acetobutylicum aad gene did not hybridize to three species of solvent-producing clostridia, including C. saccharobutylicum NCP 262. However, we have recently isolated a gene from C. saccharobutylicum NCP 262 with significant similarity to the C. acetobutylicum aad gene (unpublished results). It is located on the genome together with the other genes concerned with solvent and acid production (Fig. 5
). The bdhAB genes and the bifunctional aad gene are involved in butanol formation, and the ctfAB and adc genes convert acetoacetyl-CoA to acetone, and are therefore very likely to be found on the NCP 262 genome. However, the C. acetobutylicum probes for the genes ctfAB and bdhAB did not hybridize with the NCP 262 genome under the conditions we used. Taken together, these results suggest that there is considerable sequence divergence between certain solvent-producing genes found in the solvent-producing clostridia. Further studies are under way to identify and sequence the remaining sol cluster genes of NCP 262.
The genome size and organization of C. saccharobutylicum were compared with those of the spore-forming bacteria B. subtilis, C. acetobutylicum, C. perfringens and C. beijerinckii (Fig. 6). The genome of C. saccharobutylicum was smaller than that of C. beijerinckii but larger than the genomes of the other three spore-forming bacteria. On the basis of genome organization, C. saccharobutylicum is most similar to C. beijerinckii and C. perfringens, with the order of the genes gyrAB, groESL, spoIID, dnaKJ and recA being conserved. In addition, several rrn operons in C. beijerinckii, C. perfringens and C. saccharobutylicum are transcribed divergently from the origin of replication, as opposed to only one rrn operon being transcribed divergently to the remaining rrn operons in C. acetobutylicum and B. subtilis.
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ACKNOWLEDGEMENTS |
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Received 24 November 2001;
revised 10 March 2001;
accepted 23 March 2001.