General features of the genome and comparative genomics

The major features of the genome are listed inTable 1 andFigure 1. The genome of proteolyticC. botulinum strain Hall A (ATCC 3502) consists of a chromosome of 3,886,916 bp and a plasmid, pBOT3502, of 16,344 bp, which carry 3650 and 19 coding sequences (CDS), respectively. The %GC content (26.8%) of the plasmid is slightly lower than that of the chromosome (28.2%).

Plasmid pBOT3502, which is not similar to other sequenced clostridial plasmids, contains a large CDS (CBOP01), encoding a protein of 1194 amino acids that shows significant similarity with the alpha-subunit of DNA polymerase III (DnaE), the closest match being the chromosomally encoded DnaE ofC. perfringens. This class of protein is not normally associated with plasmid replication. Homologs have been noted on Ti-plasmids, but in these cases, additional genes encoding other, more traditional, plasmid replication proteins are also present. The sole involvement of CBOP01 in replication of pBOT3502 has been demonstrated through the subcloning of the region encompassing this gene into replicon cloning vectors (M.H.J. Wells-Bennik, K. Medendorp, A.T. Carter, and M.W. Peck, unpubl.). Other CDSs are likely to be involved in plasmid stability (CBOP05) and mobilization (CBOP11 and CBOP12), but the most prominent CDSs are those encoding a biosynthetic and transport system apparently dedicated to the production of a bacteriocin (CBOP15-19) that shares 40% identity, at the amino acid level, with a boticin of proteolyticC. botulinum strain 213B (Dineen et al. 2000). Given the CDSs present in the plasmid, the major pressure for retention of pBOT3502 by the host is likely to be the production of boticin, which may offer a competitive advantage to the strain, outweighing the metabolic burden imposed by plasmid maintenance.

Unlike the highly mosaic genome ofC. difficile (Sebaihia et al. 2006), there is no evidence of recent horizontal gene acquisition in theC. botulinum genome, apart from some selfish elements; two prophages, two prophage remnants, and 12 transposases (only one of which is intact), which are dispersed throughout the chromosome.

There is very little overall synteny between the genomes of the sequenced clostridia, confirming further the heterogeneity of theClostridium genus. To identify sets of genes that are shared or unique toC. botulinum, reciprocal FASTA analysis of theC. botulinum CDSs was performed against four sequenced clostridial genomes,C. acetobutylicum,C. perfringens strain 13,C. tetani, andC. difficile. There are only 568C. botulinum CDSs (16%) that are shared with all the other sequenced clostridia, while 1511 CDSs (41%) have orthologs in at least one, but not all, of the sequenced clostridia, and 1571 CDSs (43%) are unique toC. botulinum, compared to the other four sequenced clostridial genomes. The shared CDSs mainly encode core functions, whereas the CDSs that are unique toC. botulinum encode accessory functions (Supplemental Fig. S1). The distribution ofC. botulinum unique genes is markedly nonhomogeneous around the genome (Fig. 1); there is no readily apparent reason for this, although a remarkably similar distribution was recently described in the genome ofC. perfringens (Myers et al. 2006).

Neurotoxin genes and virulence factors

The main virulence factor ofC. botulinum, the neurotoxin, is produced as a noncovalently bound complex with two or more nontoxic protein components, hemagglutinin, and nontoxic nonhemagglutinin. To date, the phenotypic and genotypic designation of the proteins and genes involved in the production of clostridial neurotoxins (of bothC. botulinum andC. tetani) has been both unconventional and has lacked consistency. In this annotation we have, therefore, adoptedcnt (Clostridial NeuroToxin) as the prefix for genes involved in the production of botulinum, and tetanus, neurotoxins. All botulinum and tetanus neurotoxin genes arecntA. All nontoxic, nonhemagglutinin (NTNH) genes arecntB; the genes encoding hemagglutinin proteins are designated ascntC,cntD, andcntE, and replaceha34,ha17, andha70, respectively; and the gene that encodes the regulatory protein that controls their expression iscntR. The genes of the various toxins and their accessory proteins are clustered either on the chromosome (Group IC. botulinum and Group IIC. botulinum), a prophage (Group IIIC. botulinum), or a plasmid (Group IVC. botulinum). In Group I (proteolytic)C. botulinum strain Hall A (ATCC 3502), they are organized in two divergent transcriptional units on the chromosome,cntAB andcntCDE, which bracket the regulatory gene,cntR. The whole cluster is flanked on the right end by three transposases, and on the left end by a single transposase. The association of this locus with mobile elements (transposases) raises the possibility that the whole locus may be mobile, and it may have been laterally acquired, although there is no direct evidence that it has been transposed as a unit. It may also explain why strains can appear to lose the ability to form neurotoxin when repeatedly cultured in the laboratory (Lund and Peck 2000). The sequence of the neurotoxin gene and the organization of the neurotoxin gene cluster are typical of that for subtype A1 toxin, and closely resemble that of other strains (Dineen et al. 2003;Smith et al. 2005). There are no other full or partial neurotoxin genes in the genome.

Following synthesis of the neurotoxin, proteolytic cleavage of the toxin at one-third the distance from the N terminus, to produce the Heavy and Light Chains, is required for toxicity. The identity of the protease responsible is not known. One study has previously purified a 62-kDa protein from culture supernatant ofC. botulinum that is believed to carry out this proteolytic nicking (Dekleva and Dasgupta 1990). Polyacrylamide gel electrophoresis revealed that the 62-kDa protease, which is specific for the arginyl peptidyl bond, is composed of 15.5- and 48-kDa polypeptides. Interestingly, the structure and substrate specificity of this enzyme are reminiscent of those of the secreted alpha-clostripain fromClostridium histolyticum (Dargatz et al. 1993), a homolog (74% amino acid identity) of which is present inC. botulinum (CBO1920). TheC. histolyticum alpha-clostripain is a trypsin-like cysteine endopeptidase with strict specificity for the arginyl bond. It is synthesized as an inactive prepro-enzyme that undergoes an autocatalytic cleavage to generate 15.4- and 43-kDa polypeptides, which associate to form a heterodimeric active enzyme (Dargatz et al. 1993). Furthermore, both theC. histolyticum alpha-clostripain and theC. botulinum 62-kDa protease require a reducing agent and calcium for full activity and are susceptible to the same protease inhibitors. These data strongly suggest that theC. botulinum ortholog of alpha-clostripain (CBO1920) is the endogenous protease responsible for the proteolytic nicking of the neurotoxin ofC. botulinum. At this stage it is not clear whether the sole purpose of this protease is to carry out the post-translational processing of the neurotoxin, or if it also contributes to the overall proteolytic activity ofC. botulinum (see below). A gene encoding clostripain (CPE0846) is also present inC. perfringens, and has been found to be positively regulated by the two-component system VirR/VirS (Shimizu et al. 2002b).

A prominent feature of theC. botulinum genome that is absent from all the other sequenced clostridial genomes is the presence of a nine-gene cluster (CBO0486–0494) highly similar to the streptolysin S (SLS) biosynthetic operon (sagA–I) fromStreptococcus spp. The SLS is a cytolysin that is responsible for the hemolytic phenotype, and is an important virulence factor ofStreptococcus spp. (Datta et al. 2005).

CBO2038 encodes a putative secreted protein containing a thrombospondin type 3 repeat (PF02412). This domain is associated with proteins that are involved in binding to components of extracellular matrices such as fibronectin and collagen. This CDS is part of a gene cluster comprising CBO2043, which encodes a protein similar to the myosin-cross-reactive antigen fromStreptococcus pyogenes (Kil et al. 1994), orthologs of which are also present inC. perfringens (CPE0378) andC. tetani (CTC1855), but not inC. acetobutylicum andC. difficile.

Extracellular enzymes and general metabolism

Production of extracellular proteases by proteolytic clostridia has been associated with food spoilage and pathogenicity. The highly proteolytic nature of Group I (proteolytic)C. botulinum is reflected in the genome by the presence of several protease-encoding CDSs. In addition to the putative alpha-clostripain (CBO1920, mentioned above), theC. botulinum genome encodes several other proteases including six thermolysin-like metalloproteases (CBO1441–1446); a collagenase (CBO1620); two putative hemolysins (CBO1450 and CBO1589); a C-terminal peptidase (CBO3389); and a zinc-metallopeptidase (CBO3606). All these proteases appear to have an N-terminal signal sequence, which suggests that they are likely to be secreted.

Interestingly, the genes encoding the six thermolysin-like metalloproteases (CBO1441–1446) are tandemly arrayed, and the proteins they encode are highly similar (60%–80% amino acid identity) to each other. This six-gene locus is absent in all the other sequenced clostridia. Moreover, the block of DNA encompassing these protease genes displays an anomaly in the strand-specific GC bias (a bias toward G on the lagging strand, the reverse of that normally observed) (Fig. 1). Since there is no evidence that this DNA was recently horizontally acquired, the most likely explanation for this GC bias anomaly is that it is due to an inversion following a recent recombination event. It should be noted that the last CDS in this cluster, CBO1441, contains a frameshift mutation, and it is not clear if this protein is functional.

The proteinaceous products, peptides and amino acids, produced by theC. botulinum extracellular proteases can be taken up by a large number (40) of transporters. Several clostridia are able to ferment amino acids in a coupled oxidation–reduction reaction known as the Stickland reaction, in which the reduction of one amino acid (electron acceptor) is coupled to the oxidation of another amino acid (electron donor) (Stickland 1935). Analysis of theC. botulinum genome revealed the presence of fermentation pathways for several amino acids. Glycine is reduced by a glycine reductase complex (CBO1255–1264/Grd), and oxidized by a glycine cleavage system (CBO0696–699); thus it can serve both as an oxidizing and reducing agent in the Stickland reaction. Proline is reduced by a proline reductase complex (CBO2460–2490/Prd).

C. sporogenes, the non-neurotoxigenic counterpart of Group IC. botulinum, can ferment phenylalanine and leucine (Bader et al. 1982). Two gene clusters that encode key enzymes in the fermentation pathways of these two amino acids were identified in theC. botulinum genome. The first system, CBO2192–2199, is similar to 2-hydroxyisocaproyl-CoA dehydratase (HadAIBC) fromC. difficile, in the pathway of leucine fermentation (Kim et al. 2005). The second, CBO3283–3292, which is missing in the other sequenced clostridial genomes, is highly similar to phenyllactate dehydratase (FldAIBC) fromC. sporogenes, which is involved in the fermentation of phenylalanine (Dickert et al. 2002).

Although amino acids are a significant energy source for proteolytic clostridia, these organisms can also ferment sugars. Analysis of the genome revealed the presence of a large number of genes consistent with the degradation of complex polysaccharides and metabolism of a variety of sugars.

Chitin is the second most abundant polysaccharide after cellulose; it is an insoluble linear homopolymer ofN-acetyl-D-glucosamine (GlcNAc), and is the major component of invertebrate exoskeletons and fungal cell walls. TheC. botulinum genome encodes five putative secreted chitinases, CBO0728, CBO1057, CBO1081, CBO1212, and CBO2832, none of which have been found in other sequenced clostridial genomes. They all contain a catalytic domain classified in family 18 of glycosylhydrolases (PF00704), either alone or in association with additional domains such as a fibronectin type III (PF00041) and a carbohydrate-binding domain (PF02839). All but one (CBO1212) of these chitinases have an N-terminal signal sequence indicating that they are secreted into the extracellular environment (Fig. 2). One of these chitinase genes, CBO2832, is part of a gene cluster (CBO2832–2839) that is potentially involved in the transport and metabolism of GlcNAc, the product of chitin hydrolysis by chitinases. This gene is also convergently transcribed with another CDS, CBO2831, that encodes a predicted secreted protein that is weakly similar to chitinases but has no apparent catalytic or chitin-binding domains. We have also identified two additional CDSs, CBO1958 and CBO2105, that encode secreted proteins having a C-terminal chitin-binding domain, but lacking the chitinase catalytic domain. CBO1958 is also highly similar (79% amino acid identity) to another putatively secreted protein, CBO1966. However, the latter lacks both the chitin-binding and catalytic domains. Similar noncatalytic chitin-binding proteins have been found in chitinolytic organisms (Howard et al. 2003;Vaaje-Kolstad et al. 2005). Although the precise function of these proteins is unknown, it has been suggested that they may play a role in the initial interaction of the bacteria with chitin-containing surfaces (Montgomery and Kirchman 1994). In order to assess whether these proteins are involved in the degradation of chitin, we performed a chitinase assay. The presence of small zones of clearing on chitin-layered plates confirmed the ability of the sequenced Hall A strain and also proteolyticC. botulinum strain 213B to degrade chitin (Fig. 3). Chitin degradation was not, however, detected in nonproteolyticC. botulinum strain CDC 7854. Collectively, these data suggest thatC. botulinum Hall A strain has an active chitinolytic system, enabling it to use chitin as a source of carbon and nitrogen. This is not surprising considering thatC. botulinum, as a free-living organism, colonizes diverse soil and marine environments where chitin-containing organisms, such as insects, fungi, and crustaceans, are abundant.

Starch is another abundant polysaccharide that consists of a linear polymer, amylose, and a branched polymer, amylopectin. Complete degradation of starch requires the combined action of several enzymes, alpha-amylase, beta-amylase, pullulanase, and glucoamylase. Of these only one, a secreted beta-amylase (CBO1203), appears to be produced byC. botulinum. This enzyme catalyzes the removal of maltose molecules from the nonreducing ends of the starch polymers. The lack of other starch-hydrolyzing enzymes inC. botulinum suggests that this bacterium is unable to completely degrade whole starch but nevertheless can degrade starch-derived polysaccharides. This is consistent with earlier studies that showed that proteolyticC. botulinum strains were unable to hydrolyze starch in conventional tests (Dezfulian and Dowell 1980;Smith and Sugiyama 1988), yet starch and maltose could support growth (Whitmer and Johnson 1988).

Unlike some saccharolytic clostridia, proteolyticC. botulinum does not appear to have the capacity to degrade cellulose, the most abundant polysaccharide in nature.

TheC. botulinum chromosome harbors two genes that code for two putatively secreted lipases, CBO0863 and CBO2061. Lipase activity leads to the production of a thin pearly layer on and around colonies formed on media containing egg yolk, and is exploited as a simple diagnostic test for the detection ofC. botulinum (Mills et al. 1985;Lund and Peck 2000).

For uptake and phosphorylation of sugars and sugar derivatives,C. botulinum deploys 15 phosphoenolpyruvate (PEP)-dependent phosphotransferase systems (PTS), a greater number than have been found in any of the sequenced clostridial genomes with the exception ofC. difficile. TheC. botulinum PTS complement includes representatives of five of the seven known PTS families (Barabote and Saier 2005), and the individual proteins show conservation of domain structure and functional residues or motifs. The majority of phosphotransferase genes are located adjacent to genes encoding cognate enzymes for metabolism of the phosphorylated PTS products as well as transcriptional regulators, implying that expression is coordinately induced in the presence of the substrate. TheC. botulinum genome contains a single gene encoding each of the general PTS energy-coupling proteins enzyme I (CBO3438) and HPr (CBO2398), which mediate the transfer of phosphate from the donor PEP to the substrate-specific enzyme II complexes. As inC. acetobutylicum,C. perfringens, andC. tetani, but in contrast toC. difficile and the majority of other bacteria, these genes inC. botulinum are not contiguous and appear to be expressed monocistronically.

ProteolyticC. botulinum strain Hall A has a complete glycolysis pathway, but an incomplete TCA cycle.C. botulinum appears to be capable of both acidogenic (production of acetate and butyrate) and solventogenic (production of butanol and ethanol) fermentations. Interestingly, there are two copies each of the genes encoding phosphotransbutyrylase (Ptb) (CBO3118 and CBO3427) and butyrate kinase (Buk) (CBO3426 and CBO3428), which constitute the butyrate formation pathway. UnlikeC. acetobutylicum,C. botulinum is unable to produce acetone because it lacks the gene (adc) that encodes the terminal enzyme (acetoacetate decarboxylase) in the acetone formation pathway.

Components of the cell surface

Bacteria exhibit different cell surface structures according to the environmental niches they inhabit. These components play an important role in sensing and surviving environmental challenges and interaction with other organisms. Therefore, the characterization of the cell surface components ofC. botulinum and comparison with other sequenced clostridia could provide an insight into their lifestyles and the environmental niches they colonize.

The chemotaxis process allows motile bacteria to sense environmental cues and to respond by moving toward attractants and away from repellents. Methyl-accepting chemotaxis proteins (MCP) play a key role in this process. MCPs have membrane-associated N-terminal sensor domains that bind to attractants and repellents (Scott et al. 1993) and intracellular methyl-accepting signaling domains that transduce the external signal to the intracellular chemotaxis machinery (Kim et al. 1999).C. botulinum encodes 24 putative MCPs, only one of which, CBO3558, designated SonO, has been characterized. SonO was recently identified as a sensor of nitric oxide (NO), which is extremely toxic toC. botulinum, and the protein crystal structure has been solved (Nioche et al. 2004).C. botulinum encodes more MCPs thanC. tetani (15),C. difficile (1), andC. perfringens (0), but fewer thanC. acetobutylicum (38), suggesting thatC. botulinum andC. acetobutylicum have a relatively elaborate sensing capability, and can respond to a wider range of environmental cues (attractants and repellents) relative toC. tetani,C. difficile, andC. perfringens.

TheC. botulinum genome carries 84 putative chemotaxis- and flagella-related proteins, 54 of which are predominantly organized into two operons (CBO2637–2666 and CBO2730–2753). Overall, the gene content and order in these two operons is conserved betweenC. botulinum,C. acetobutylicum, andC. tetani, but to a lesser extent inC. difficile. In contrast,C. perfringens lacks all the flagellar and chemotaxis genes, which is consistent with it being defective in flagellar-mediated motility. The remaining 30C. botulinum chemotaxis- and flagella-related CDSs are dispersed, singularly or in pairs, throughout the chromosome.

C. botulinum possesses five CDSs that putatively encode the structural subunits of the flagellar filament (CBO0242, CBO2666, CBO2695, CBO2730, and CBO2731), more than inC. acetobutylicum (4),C. tetani (4),C. difficile (1), andC. perfringens (0).C. botulinum has an additional CDS, CBO0798, that is similar to only the N-terminal domain of a flagellin structural subunit.

The two major flagellar and chemotaxis gene clusters ofC. botulinum are separated by two gene clusters (CBO2678–2689 and CBO2696–2729) that potentially encode proteins involved in the biosynthesis, modification, polymerization, and export of polysaccharides. One of these clusters, CBO2696–2729, is flanked on the right by two almost identical (98.9% amino acid identity) flagellin structural genes, CBO2730 and CBO2731, that have been recently identified as the genes encoding the major structural components of the flagellar filament (Paul et al. 2007); and on the left by a third flagellin gene (CBO2695). The colocalization of this polysaccharide biosynthesis locus and the flagellin structural genes is similar to that of the flagellar glycosylation locus inCampylobacter jejuni (Szymanski et al. 2003).

Polysaccharide biosynthesis loci, located at an equivalent location to that ofC. botulinum, were also identified inC. acetobutylicum andC. tetani, CAC2168–2202 and CTC1692–1714, respectively, but not inC. perfringens (defective in flagellar-mediated motility). However, although these loci share a few genes at their 5′-ends, the majority of their genes are either different or highly divergent, suggesting that they produce different glycan structures. InC. difficile only the ortholog (CD0240) of the first CDS of theC. botulinum polysaccharide biosynthesis cluster (CBO2729), which encodes a glycosyltransferase, is present. TheC. acetobutylicum andC. tetani polysaccharide biosynthesis clusters and the glycosyltransferase (CD0240) ofC. difficile also lie directly downstream from one flagellin gene, CAC2203/flaC, CTC1715, and CD0239/fliC, respectively.

Interestingly, motility accessory factors (MAF) of the Cj1318 family were also identified within the polysaccharide biosynthesis loci ofC. botulinum (CBO2728),C. acetobutylicum (CAC2168, CAC2196, and CAC2202), andC. tetani (CTC1697 and CTC1714). In addition toCampylobacter, members of the Cj1318 family have only been identified in the genomes ofHelicobacter, Treponema, andLeptospira; all of which were reported to modify their flagellins with glycan (Wyss 1998;Schirm et al. 2003).

The location of theC. botulinum,C. acetobutylicum, andC. tetani polysaccharide biosynthesis loci adjacent to the flagellar operon and the similarity in both gene content and organization to the known flagellar glycosylation locus ofC. jejuni strongly suggest that these loci are most probably involved in the glycosylation of flagellins in these clostridial species. This view is supported by several lines of evidence: (1) The whole locus as well as the flagellar operon are missing inC. perfringens. (2) The modification ofC. acetobutylicum flagellin protein, CAC2203/FlaC, is sensitive to neuraminidase treatment, suggesting it is glycosylated with a sialic-acid-like sugar (Lyristis et al. 2000). (3) The flagellin ofC. difficile has been reported to be post-translationally modified (Tasteyre et al. 2000), and the modification is likely to be glycan, however, with significantly fewer putative glycosylation genes associated with the flagellin genes. The modification probably differs from that ofC. botulinum,C. tetani, andC. acetobutylicum; (4) Flagellin glycosylation has also been reported inClostridium tyrobutyricum (Arnold et al. 1998).

C. botulinum has a second gene cluster that is also potentially involved in the biosynthesis of a surface polysaccharide structure, CBO3092–3114. BothC. acetobutylicum (CAC2310–2337) andC. tetani (CTC2252–2270) have similar loci at an equivalent location to that ofC. botulinum. However, putative polysaccharide biosynthesis loci inC. perfringens (CPE0613–0629) andC. difficile (CD2767–2801) are located elsewhere in their genomes. In addition, the genome ofC. perfringens carries another gene cluster, CPE0461–0511, which is absent in all the other sequenced clostridia, and which potentially encodes the necessary functions for capsule biosynthesis. Among the sequenced clostridia, the presence of a capsule-like structure has been reported only inC. perfringens (Sheng and Cherniak 1997) andC. difficile (Davies and Borriello 1990;Baldassarri et al. 1991).

Type IV pili are important for the tight adhesion of bacteria to host cells and solid surfaces and mediate bacterial twitching motility. All the sequenced clostridia appear to produce type IV pili as indicated by the presence of gene clusters that encode the necessary functions for the assembly of this cell surface appendage; CBO1900–1909, CAC2096–2105, CPE1836–1844, CTC1595–1604, CD3290–3297, and CD3503–3513, inC. botulinum,C. acetobutylicum,C. perfringens,C. tetani, andC. difficile, respectively. The prepilins encoded in the sequenced clostridia exhibit similarities to type IV prepilins (Craig et al. 2004). Among the sequenced clostridia, the presence of pili has been previously reported inC. difficile (Borriello et al. 1988) and more recently inC. perfringens (Varga et al. 2006).

S-layer proteins form crystalline arrays on the surface of numerous bacteria and archaea (Sara and Sleytr 2000). A small region of the genome was identified that contained CDS that encode several putative S-layer proteins (CBO00374, CBO0378, CBO0379, and CBO0380). These proteins all contain three cell wall binding domains, the role of which is to anchor proteins to the cell surface, and are similar to the S-layer protein, SlpA, ofC. difficile (Calabi et al. 2001). The amino acid compositions of CBO0378 and CBO380 are comparable to that determined for an antigenic cell wall protein of proteolyticC. botulinum type A strain 190L (Takumi et al. 1983), suggesting that these genes may encode the S-layer proteins for Hall A.

Conclusion

Analysis of theC. botulinum genome revealed novel and interesting aspects of its lifestyle. The genome supports the view ofC. botulinum as an essentially saprophytic organism that uses its toxin to rapidly kill a host for subsequent saprophytic utilization. The fact that it does not spend long periods associated with a living host may explain its relatively stable genome, contrasting with that ofC. difficile, which can spend long periods coexisting with its host, and has a highly variable genome correlating with this highly dynamic niche.C. botulinum is both a proteolytic and chitinolytic bacterium, producing several extracellular proteases and chitinases. The combined action of the adhesins, extracellular matrix-binding proteins, proteases, and cytolysin may contribute to the softening and extensive destruction of tissues of rotting carcasses of dead animals; whereas the chitinolytic system may be deployed to degrade chitin-containing invertebrate species such as insects, fungi, and crustaceans.C. botulinum may well deploy the plasmid-encoded boticin to defend this nutrient-rich source against different microbial competitors. In addition to metabolism of peptides and amino acids, the bacterium has a significant capacity for uptake and metabolism of sugars and related molecules.

Like many of the clostridial genome projects before it, the data generated have provided a fascinating insight into the physiology of this pathogen. Exploitation of this information in hypothesis-driven research has, however, until now been severely impeded by a general absence of clostridial mutational tools for functional genomic studies. This deficiency has recently been solved through the generation of a gene knockout system forC. difficile (O’Connor et al. 2006) and a new system that has been shown to be applicable to a range of clostridial species (N.P. Minton, unpubl.). The availability of such systems will considerably aid future analysis of clostridial genomes.

Methods

ATCC 3502 is one of the most widely studiedC. botulinum strains. It was deposited at the ATCC in 1940 by I.C. Hall, and is listed as Hall Strain 174. This strain belongs toC. botulinum Group I (proteolyticC. botulinum) and forms type A1 neurotoxin. The Health Protection Agency Center for Emergency Responsiveness and Preparedness at Porton Down (formerly the Center for Applied Microbiology, CAMR) obtained this strain from the ATCC in 1987 as freeze-dried ampoules from Batch MED-38 (prepared in 1981). The strain has been deposited with the NCTC as NCTC 13319.

Cloning and sequencing

The initial genome assembly was obtained from 69,632 paired end sequences (giving 9.15-fold coverage) derived from four genomic shotgun libraries (all in pUC18 with insert sizes of 1.5–2.0 kb and 2.0–2.2 kb, 2.2–2.5 kb, and 2.5–4.0 kb) using dye terminator chemistry on ABI3700 automated sequencers; 1604 paired-end sequences from one pBACe3.6 library with insert sizes of 15–23 kb (a clone coverage of 3.9-fold) were used as a scaffold. A further 9343 directed sequencing reads were generated during finishing.

Sequence analysis and annotation

The sequence was assembled, finished, and annotated as described previously (Sebaihia et al. 2006), using Artemis (Rutherford et al. 2000) to collate data and facilitate annotation. The DNA and predicted protein sequences ofC. botulinum were compared to the sequenced clostridial genomes using the Artemis Comparison Tool (ACT) (Carver et al. 2005). Orthologous gene sets were calculated by reciprocal best-match FASTA comparisons with subsequent manual curation. Pseudogenes had one or more mutations that would prevent translation; each of the inactivating mutations was checked against the original sequencing data. Horizontally acquired DNA was identified by anomalies in G+C content and GC deviation, combined with the presence of mobility elements such as transposases and integrases. All such regions were subject to manual checking and curation.

Microarray design

A 100–500 bp probe for each CDS was produced by PCR using genomic DNA ofC. botulinum Hall A (ATCC 3502) as template. PCR products were checked by agarose gel electrophoresis and purified by precipitation with isopropanol. Array printing buffer was 0.3× saline sodium citrate, 50% dimethyl sulfoxide (DMSO). The probes were spotted onto gamma amino propyl silane-coated GAPS II slides (Corning) using a Stanford arrayer (Thompson et al. 2001). Owing partly to the very low G+C content and to multiple-copy CDS, several loci failed primer and/or predicted PCR product specificity analysis (assessed using BLAST). It was only possible to include probes for 3433 CDS (out of 3650 predicted, ∼94%). The array also included a probe for each CDS identified for the plasmid pBOT3502.

Preparation of genomic DNA

Genomic DNA was purified from exponentially growing cells by lysis in lysozyme/mutanolysin, incubation in proteinase K/SDS, followed by a standard phenol/chloroform extraction procedure. Final pellets were dissolved in TE (10 mM Tris-HCl at pH 8.0, 1 mM EDTA). Genomic DNA was digested to completion with Sau3A1 (Promega) and purified by phenol/chloroform extraction. Genomic DNA was labeled overnight (∼14 h) with Cy5-dCTP or Cy3-dCTP fluorescent nucleotides (Amersham) using a BioPrime Array CGH kit (Invitrogen) and purified for hybridization using QiaQuick PCR cleanup columns (QIAGEN).

Microarray hybridization and data analysis

For each experiment, 2 μg of Cy5-labeled Hall A (reference) DNA and 2 μg of Cy3-labeled test DNA were hybridized onto the array under a glass coverslip in a humidified dual microarray hybridization chamber (Monterey Industries) overnight at 54°C. DNA microarrays were then scanned using an Axon GenePix 4000A microarray laser scanner (Axon Instruments) using GenePixPro 5.1 software. All data were subsequently analyzed using the R statistical language and environment (http://www.r-project.org), specifically with the tools available from the Bioconductor Project (http://www.bioconductor.org), predominantly the LIMMA Bioconductor package (Smyth 2004;http://www.bioconductor.org/packages/bioc/1.9/html/limma.html). Fluorescence data were background-subtracted using a Bayesian model-based algorithm (Kooperberg et al. 2002), then normalized within each array, using “printtiploess” to correct for spatial and other artifacts. Scatterplots and/or box plots of the raw and normalized fluorescence values were manually inspected prior to proceeding with linear model fitting, according to the appropriate design matrix, with empirical Bayesian smoothing/moderation of the standard errors. The linear modeling was output as toptables, comprising log₂-based coefficients (test vs. reference fold ratios), with appropriate significance statistics for each locus on the array in each genome tested. Coefficients were imported into GACK (Kim et al. 2002), and a trinary analysis was performed to assign present/uncertain/absent or diverged status to all loci in all samples. EPP thresholds of 0% and 100% were applied. GACK output was plotted as a heatmap, with loci ordered by physical location in theC. botulinum (Hall A) genome.

Chitin degradation assay

The ability to degrade chitin was determined by spottingC. botulinum cultures onto layered peptone-yeast extract-glucose-starch (PYGS) agar plates (Stringer et al. 2005), in which the upper layer additionally contained 2% finely ground chitin. Clearing was assessed after incubating for 8 d at 30°C under a headspace of H₂/CO₂ (90/10). Cell extract from the chitinolytic bacteriumStreptomyces griseus was included for comparison.

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