Introduction

Clostridium botulinum strains have been classified by the Center for Disease Control and Prevention as “Category A agents” with highest—risk threat especially for bioterrorism [1, 2]. Botulinum neurotoxins (BoNTs) produced by C. botulinum are extremely lethal, such that 3 g are sufficient to kill the entire population of United Kingdom, and 400 g to wipe out the whole mankind [1, 3]. The need is to identify molecular markers for distinguishing closely related strains [2, 4]. Bacterial identification through sequence analysis of 16S rRNA gene (rrs) has been widely exploited [5, 6].

Challenges in identification of C. botulinum are the variations arising largely due to the different types (A–G) of neurotoxins [7, 8]. Phylogenetic lineages are of 4 types: (1) Group I—proteolytic C. botulinum types A, B and F, and C. sporogenes, (2) Group II—nonproteolytic types B, E and F, (3) Group III—types C and D and C. novyi type A, and (4) Clostridium argentinense (C. botulinum type G), related to C. subterminale [7, 9, 10]. BoNT toxin is encoded by the bont gene. Another feature of genetic variability among C. botulinum, is the multiple copies of rrs in each genome (http://rrndb.umms.med.umich.edu/search/). Recent studies have shown certain unique features within rrs, such as 30–50 nts signatures and Restriction endonucleases (RE) digestion patterns. These can be exploited to identify organisms [5, 6, 11, 12]. In silico RE digestion patterns with an array of these enzymes revealed variations in these sites yielding unknown profiles. Of the various REs used in our previous study involving 128 rrs sequences of C. botulinum, RE-BfaI digestion pattern—5′ 195-20-163-29-146-185-331 (nts) 3′ was reported to be unique. It was thus proposed for usage as a molecular marker for species level identification [6]. However, certain naturally occurring genetic variations in RE sites were observed in closely related strains. These modifications were observed due to changes in the nucleotides of the RE recognition sites. Two questions were raised (1) Can we use these genetic changes as marker to distinguish closely related strains? and (2) What is the evolutionary significance of these nucleotide changes (NC) within RE sites.

Materials and Methods

rrs-Genome Sequence Data Collection

Information on the rrs of 13 completed genome sequences of C. botulinum used in this study was downloaded from Ribosomal Database Project II (http://rdp.cme.msu.edu/genome/) (Table S1). The genome sizes of different strains of C. botulinum were obtained from REBASE Genomes (http://tools.neb.com/~vincze/genomes/), their GC contents (% mol) were retrieved from NCBI database (http://www.ncbi.nlm.nih.gov/genomes/MICROBES/microbial_taxtree.html) and the copy numbers of rrs within the complete genomes were obtained from rrndb database (http://rrndb.umms.med.umich.edu/search/) [13, 14]. The GC % of each rrs was calculated for all C. botulinum genomes using BioEdit [17] (Table S1).

Analysis of Intra- and Inter-genomic Heterogeneity

A comparative study of intra- and inter-genomic heterogeneity in rrs of 13 completely sequenced genomes of C. botulinum was performed by multiple sequence alignment using Clustal X version 2.0.12 followed by Data Analysis in Molecular Biology and Evolution (DAMBE) software Package [16, 17]. It enabled us to bring down the redundancy among the 212 copies of rrs of 13 different strains (Tables S1 and S2) to 130 representatives: (1) 130 copies of rrs could be reduced to 49 representatives due to 100 % similarity among them, and (2) 81 rrs copies showed distinct heterogeneity. Hence, all subsequent analyses were based on these 131 representative copies of rrs.

Heterogeneity Analysis of rrs

Intragenomic heterogeneity among all the rrs sequences within each of the 13 genomes of C. botulinum strains was analysed by multiple sequence alignment (Clustal X version 2.0.12): (1) between non substituted rrs sequence (Okra, S001014409) and others and by counting the nucleotide changes (NC) with the help of BioEdit, and (2) pairwise alignment between two rrs within the strain and by counting NC using BioEdit (Table S1) [15, 16].

For the intergenomic heterogeneity, non substituted rrs sequences of 13 different C. botulinum strains were aligned by multiple sequence alignment (Clustal X version 2.0.12) and a completely non-substituted representative rrs sequence (Okra, S001014409) of C. botulinum was identified using BioEdit (Table S1) [16, 17]. The intergenomic heterogeneity was calculated by counting NC in the reference sequence which was chosen from representative sequences with the help of BioEdit [16, 17].

Restriction Endonuclease Analysis

A total of 241 Type II REs consisting of 4–6 nts cutters and larger recognition sites (>6 nts) listed in BioEdit [15] were considered for these analyses. Since 104 REs proved to be non-cutters, only 137 REs were used for further analyses (Table S3). Then we concentrated on those RE sites which were common to all the strains and especially those where NCs led to changes in RE sites. Thus only those nucleotides which affected the RE sites were considered for data matrix. Consensus RE patterns—frequency of occurrence of RE sites and the pattern of fragments (nts) were determined for each rrs by employing: (1) 4 nts cutters—BstUI(CG’CG), HpyCH4V(TG’CA), TaqI(T’CGA), and Tsp509I(‘AATT), (2) 5 nts cutters—HpyCH4III(ACn’GT), Hpy188I(TCn’GA), and Tsp45I(‘GTsAC);and (3) 6 nts cutters—NlaIV(GGn’nCC) and Hpy188III(TC’nnGA) [5] (Tables S4, S5, S6).

Analysis of Nucleotide Changes

NCs leading to changes in RE sites in rrs genes of different C. botulinum genomes were detected by comparing them with C. botulinum strain Okra rrs: S001014409 as reference (Table S1). NCs in RE sites within rrs were categorized as: (1) appearance of distinct ones, (2) creation of additional RE sites, (3) loss of RE sites, and (4) no evident change in RE sites (Tables 1, 2, S7, S8, S9). In addition, certain NCs were also observed in the non-RE sites in all the strains.

Table 1 Occurrence of distinct restriction endonuclease sites in rrs of Clostridium botulinum strain
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Table 2 Evolution of additional restriction endonuclease sites due to nucleotide changes in rrs of Clostridium botulinum strains
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Phylogenetic Analysis

For phylogenetic analysis, rrs sequences from all the 13 C. botulinum strains and their close phylogenetic associates as reported in literature [2, 8, 18] like C. argentinense, C. beijerinckii, C. butyricum, C. haemolyticum, C. novyi, C. proteolyticus, C. schirmacherense, C. sporogenes and C. subterminale were assembled and aligned using the multiple alignment program Clustal X version 2.0.12 [16]. In order to estimate evolutionary distance, pairwise distances between all species were calculated with the DNADIST of the PHYLIP 3.69 package [5, 19]. The resultant distance matrix was then used to draw a neighbour joining tree with the program NEIGHBOR [19]. The program SEQBOOT [19] was used for statistical testing of the trees by resampling the dataset 1,000 times. The trees were viewed through TreeView Version 1.6.6 [20]. A phylogenetic tree of 130 rrs sequences of C. botulinum and their close associates was drawn to check if these 131 rrs sequences show closeness exclusively to BoNT producers—C. argentinense, and C. butyricum, or not. We did not observe any discrepancy in the phylogenetic relationships of C. botulinum strains with those reported in literature (Fig. S1).

Cladistic Analysis

For cladistic analysis, nt sequences of 38 HKGs (Table S10) (abrB, arcA1, atpA, atpC, bofA, ccpA, cphA, cphB, dltB, dltD, dnaA, dnaN, gyrA, gyrB, ispE, ispF, ksgA, mdeA, metG, mnaA, nrdD, nrdG, prfA, psd1, psd2, pyrG, recF, recR, Rho, rpiB, rpmE, serS, sfsA, spmA, spmB, tagO, tdK, and Upp) of 13 C. botulinum strains were retrieved from NCBI (http://www.ncbi.nlm.nih.gov/genomes/MICROBES/microbial_taxtree.html) in FASTA format. For cladistic analysis, initially trees for all the 38 genes were drawn. Six (dnaN, gyrB, metG, prfA, pyrG, and Rho) out of these 38 genes were selected on the basis of the high frequency of the close relationship between 2 and 3 strains and compared them with the relationship recorded with rrs.

For the final presentation of Cladistic analysis, seven concatenated HKGs including rrs of C. botulinum strains (Table S1 and S10) were assembled and aligned using the multiple alignment program Clustal X version 2.0.12 [16], and saved in NEXUS format. The maximum parsimony tree was constructed with from the PAUP* (Phylogenetic Analysis Using Parsimony) ver. 4.10b [21] using heuristic search methods and bootstrap test with 100 replicates (Fig. 1).

Fig. 1
figure 1

Maximum parsimony (MP) tree for the concatenated six HKGs with rrs. Most-parsimonious tree generated from the concatenated gene sequences of pyrG, gyrB, dnaN, metG, prfA, and RhO (total length 21113–22616 bp) showing the relationship between the 13 Clostridium botulinum strains. MP analysis was performed using Paup* ver. 4.0b10

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Results

The availability of thirteen completely sequenced genomes of C. botulinum of diverse geographic origins with high microheterogeneity (9–22 copies of rrs per genome) (Table S1) [2] prompted us to use them as a model system for exploring unique molecular markers in their rrs gene sequences. Comparisons of rrs sequences within and between genomes revealed that the most heterogenous rrs sequences were observed within C. botulinum strains ATCC3502, ATCC19397/Hall, H04402065, Kyoto, Langeland and Okra (Table S2). In silico mapping with different type II REs allowed us to establish the extent of variability in digestion patterns in rrs due to NC (Tables S4, S5, S6).

Variability in RE Digestion Patterns of rrs

Genetic evidence on the origin of the 13 different strains of C. botulinum was established through in silico mapping of rrs with the following REs: BstUI, Hpy188I, Hpy188III, HpyCH4III, HpyCH4V, NlaIV, TaqI, Tsp45I, Tsp509I (Tables S4, S5, S6). In silico RE mapping with Tsp509I resulted in different digestion patterns, each having 3 fragments of distinct lengths (Table S6): (1) 244-40-46 nts, (2) 556-244-40 nts, (3) 461-244-40 nts, and (4) 644-244-40 nts. The appearance of 244-40 nts fragments in all copies of rrs of 12/13 strains of C. botulinum provides potential evidence of their common origin. This proposal was supported by similar observations with different REs: (1) BstUI (166-100-445), (2) Hpy188I (854-56), (3) Hpy188III (96-88-338-275), (4) NlaIV (1-524), and (5) HpyCH4III (261-149 nts) (Tables S4, S5, S6). C. botulinum strain BKT015925 was quite distinct from the rest 12 strains. Only one copy of rrs of strain BKT015925 showed resemblance with respect to digestion pattern obtained with Tsp509I to those from Kyoto (1/18 copies), 657 (2/18 copies) (Table S6). On the other hand, the strain BKT015925 resembled Alaska and Eklund 17B with respect to the digestion patterns obtained with RE BstUI (Table S5).

Variability in the fragments observed at 5′ end of rrs sequences with RE -Hpy188I, at 3′ end with RE -BstUI, and at both the ends with RE -HpyCH4III and -TaqI provided evidence for the presence of unique features in these strains. These observations allowed us to segregate the 13 strains of C. botulinum into 2 major groups (Tables S4, S5, S6). The first group composed of Alaska E43 and Eklund 17B, showed exactly similar digestion patterns in all their 22 rrs copies with three different REs: (1) Hpy188III—5′ 502-96-88-261-76-274-86 (nts) 3′, (2) HpyCH4V—5′ 309-22-217-25-395-204 (nts) 3′, and (3) Tsp509I—5′ 244-40-46 (nts) 3′ (Tables S5, S6).

Evidence of co-evolution of Kyoto and 657 and subsequent independent evolution of Kyoto from the second group of 8 C. botulinum strains, can be proposed on the basis of their sharing of RE patterns for—HpyCH4V and Tsp509I. C. botulinum strain Kyoto can be separated from 657 due to unique RE digestion patterns in the former: (1) RE-HpyCH4V 5′ 133-415-25-346-49-46-215 (nts) 3′ (Table S5), and (2) RE-Tsp509I 5′ 461-244-40 (nts) 3′ (Table S6). Similarly, unique RE digestion patterns can be used to identify the following: (1) ATCC 19397/Hall by (a) Hpy188I—5′ 70-267-856-56 (nts) 3′ (Table S6), (b) Hpy188III—5′ 308-196-96-88-337-274 (nts) 3′ (Table S5), and (2) ATCC3502 by (a) Hpy188III—5′ 249-196-96-88-337-275 (nts) 3′ (Table S5), and (b) TaqI—5′ 90-784 (nts) 3′ (Table S4).

Novel Markers for Identification of C. botulinum Strains

Details of the variability in the fragmnents obtained as a result of digestion of rrs of C. botulinum with REs—TaqI, Hpy188III, Tsp509I, HpyCH4III, Hpy188I, NlaIV, HPYCH4V and Tsp45I and their use as potential markers has been presented as supplementary material (Tables S4, S5, S6). In summary, the various observations confirmed the previous findings that these 13 C. botulinum strains have been grouped as (1) proteolytic and (2) non-proteolytic (Alaska E43 and Eklund 17B) [18]. In addition, it may be emphasized that the method employed in this study was able to not only differentiate between group I and group II strains, but could also differentiate individual strains, at least within group II.

The most striking feature which emerged from this in silico RE activity was the variation in the number of fragments in different copies of rrs gene of a given C. botulinum strain. With RE-BstUI, 22/22 copies of rrs of Alaska E43, 14/22 copies of Eklund 17B and 8/10 copies of BKT015925 showed a pattern of 166-100-445-200-94-87-15 nts (Table S5). In the second set of 8/22 copies of Eklund 17B and 2/10 copies of BKT015925, the pattern changed to 166-100-444-200-180-15 nts, which could occur by the merger of 94-86 nts fragments into a single fragment of 180 nts. In the rest of the 8 C. botulinum strains, the the merger of the fragments 200-94-86 or 200-180 seem to have given rise to a much bigger fragment of 380 nts (Table S5). Such cases of disappearance of RE sites leading to fewer but larger fragments were observable also with other REs (Tables S4, S5). These observations led us to trace the evolutionary fate of the RE sites which perhaps disappeared primarily through natural genetic mutations.

Nucleotide Changes

Multiple alignments of all copies of rrs from different C. botulinum strains enabled us to trace the sequence of events of NCs occurring within the rrs gene sequences. In each C. botulinum genome, 2–6 out of 9–22 rrs copies did not show any NC. Further multiple alignments among these rrs with no NC enabled us to identify a sequence which could be categorized as a potential candidate for a “common ancestor” to these 13 different strains of C. botulinum. This “ancestral” rrs (S001014409) sequence belonging to C. botulinum strain Okra was devoid of any NC (Table S1). It served as reference rrs sequence for elucidating the fate of NC in RE sites, in the following 4 forms: (1) appearance of distinct sites, (2) gain of additional sites, (3) loss of sites, and (4) no evident change. In this study, we did not attempt to elucidate the significance to NC in non-RE sites observed in these C. botulinum strains.

Evolution of Distinct RE Sites

Changes in nucleotides within RE sites in rrs sequences leading to distinct RE sites were observed in 10 C. botulinum strains: 657, Loch Maree, Eklund 17B, ATCC 3502, ATCC 19397, Kyoto, Langeland, 230613, H0440265 and BKT015925 (Table 1; Fig. 2). The unique features emerging from the different NC were: (1) the number of rrs copies affected within a strain varied up to 6, the most affected being those belonging to 657, Eklund 17B, ATCC 3502, and ATCC 19397, (2) the number of affected sites were 2 per rrs copy in cases of Eklund 17B, and ATCC 19397 and 3 in case of ATCC 3502, (3) similar changes at same positions such as 454 in rrs copies of different strains—ATCC 19397, Kyoto, Langeland, H04402065 and 230613 indicating possible common origin.

Fig. 2
figure 2

Mapping of single nucleotide changes (SNC) in restriction endonuclease site(s) in rrs of Clostridium botulinum strains. No SNCs affecting RE sites were observed in the 500–900 nucleotide region of the rrs

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A NC A>G in rrs of C. Botulinum 657 and Loch Maree had a dramatic impact on the RE sites. Here, 3 and 10 distinct RE sites were generated at positions 1,420/1,427 and 222/229, respectively, in strain 657. On the other hand, similar transition of A>G resulted in 4 distinct RE sites at position 1,319/1,326 in Loch Maree (Table 1). Similar observations were also made in the rest of the 3 strains of this group. The mutations of G>A in rrs: (1) S001094720 of C. botulinum Eklund 17B modified Hpy188I–TCn’GA to BstBI–TT’CGAA and TaqI–T’CGA. Transition of C>T affected three C. botulinum strains ATCC 3502, ATCC 19397 and Eklund 17B. Here, 6 RE sites were found to have evolved into 6 distinct RE sites. In addition, it was observed that RE-AciI got transformed into HpyCH4IV in ATCC 3502 and into HpyCH4III in strain Eklund 17B. In contrast to C>T, a reversal of T>C was found to influence 6 strains: ATCC 3502, ATCC 19397, Kyoto, 230613, H04402065 and Langeland. In all these 6 strains the same RE—BfaI site C’TAG was affected with slightly different out puts. This suggests a common origin of these 6 strains.

Evolution of Additional RE Sites

Appearance of additional RE sites due to NCs were recorded at different positions along the entire length of the rrs gene (Table 2; Fig. 2). Most of the mutations were transitions: (1) G>A at positions—52, 78, 79, 84, 85, 86, 87, 91, 993 and 999; (2) A>G at positions 67, 73, 74, 80, 174 and 181; (3) T>C at positions 82. 89, 171 and 178; and (4) C>T at positions 1,000, 1,006, 1,193 and 1,220 and 1,227. These NCs resulted in the generation of additional RE sites: (1) G>A lead to the generation of RE sites for Hpy188I–TCn’GA, Hpy188II–ITC’nnGA, Hpy188III–TC’nnGA, Tsp509I-‘AATT, AleI–CACnn’nnGTG, MaeIII–’GTnAC, MslI–CAynn’nnrTG, and Tsp45I–’GTsAC; (2) A>G lead to the formation of sites for activities of REs: Alu I-AG’CT, CviJI-rG’Cy, HindIII–A’AGCTT, BslI–CCnnnnn’nnGG, and EcoNI–CCTnn’nnnAGG; (3) T>C resulted in the evolution of the sites for REs: AciI–C’CGC and AciI–G’CGG; (4) C>T transition resulted in the evolution of the sites for REs: HphI–GGTGAnnnnnnnn’, and HpyCH4V–TG’CA. It may be noted here that in C. botulinum strain Okra, distinct RE sites for FatI–’CATG and NlaIII–CATG’, appeared due to simultaneous changes at positions 171T>C and 174A>G. It may be further remarked that none of these NCs was due to transversions (Purine ↔ Pyrimidine).

The impact of the appearances of additional RE sites was most evident among 4–8 rrs copies of C. botulinum strains ATCC 3502, ATCC 19397, Alaska E43) and Loch Maree. This group was followed by strains 657 and Okra, where 4 rrs copies gained RE sites (Table 2). The least affected strains were Kyoto and Langeland where gain in RE sites was evident only in a single copy each of rrs. Eklund 17B was the only strain where no gain in RE sites was recorded.

A detailed scrutiny based on the appearance of additional and distinct RE sites or loss, within rrs of different strains provided insights into their origin. The simultaneous appearance of additional RE sites in multiple copies within a strain and among different strains provided further evidence for common origin and of duplication events in these C. botulinum strains. An evidence of common origin of ATCC3502, Langeland, Loch Maree, H04402065 and Okra was obtained on the basis of transition 1220/1227C>T taking place simultaneously in 1–3 copies of rrs of each of the 4 strains (Table 2). Similarly, a transition 84/91G>A in a copy each of strain 657 and Kyoto indicate their possible common origin. Transition events in multiple copies within a strain were recorded as (1) 52/86G>A in 8 copies of ATCC 3502, (2) 67A>G in 2 copies within Loch Maree and one copies of H04402065, (3) 78/79/86G>A in 8 copies of ATCC 19397, (4) 993/999G>A and 1000/1006C>T in 8 copies of Alaska E43 and (5) 1220C>T in 2 copies of Okra, which provide support to duplication events (Table S7).

A transition G>A at positions 67/68 in 8 rrs copies belonging to 5 different strains—ATCC 19397, Eklund 17B, 230613, H04402065 and Langeland resulted in the loss of sites for 3 REs—AluI, CviJI and HindIII (Table S8). Similarly, A>G transition at position 435 was found to result in simultaneous loss of site for RE-BfaI in 6 copies of rrs belonging to 4 different strains –ATCC 19397, Kyoto, 230613 and Langeland (Table S7). These evidences support common origin of these 6 strains and subsequent diversification in Eklund 17B and Kyoto. A single transition affecting loss of RE site in multiple copies of rrs at the same position within a strain supports duplication events in the following cases: (1) 40G>A in 2 copies in ATCC 3502, (2) 67/68/74G>A in (a) 2 copies of Langeland, (b) 6 copies of ATCC 19397 and Eklund 17B each, (3) 254/261C>T in 4 copies of 657, (4) 408A>G in 3 copies of ATCC 3502, (5) 435/442A>G in 8 copies of ATCC19397 (Table S8).

Further support to the evolutionary events of common origin and duplication events was also recorded with NC in rrs leading to the generation of distinct RE sites (Table 1). A single transition event of 454/461T>C was observed in 12 rrs sequences belonging to 5 different C. botulinum strains ATCC 19397, Kyoto and Langeland, 23063 and H04402065. Here the simultaneous effect on RE-BfaI site CTAG can be explained by a common origin of these rrs sequences. Another similar T>C NC affecting RE-BfaI at position 427/461 in 6 copies of rrs of ATCC 3502 can be explained by a duplication event.

A summary of all these effects of NCs (Fig. 2) on RE sites lead us to conclude that 230613, Kyoto, Langeland, and H04402065 had a common origin, which later on diversified into Loch Maree on one hand, most probably from Kyoto and on the other hand ATCC 19397/Hall, originated from H04402065. It is also quite evident that Langeland might have given rise to Loch Maree and Okra. Redundancy in different copies of rrs within a strain was evident due to simultaneous NC in multiple copies such as (1) 4 NC events at positions 40, 52, 408 and 427 in 2–4 copies within ATCC 3502, (2) 3 NC events at positions 67, 78/79, and 435 in 3–4 copies of ATCC 19397, (3) simultaneous NC at positions 993 and 1,000 in 4 copies of Alaska E43, and (4) in 2–3 rrs copies each of strains 657, Eklund 17B, Langeland, Loch Maree and Okra.

Discussion

Genetic and functional variation due to NCs (transitions or transversions) within the coding regions may cause amino acid exchange or even lead to stop codons. In the non-conservative region, these may influence structure and/or function of the protein [22]. In general, NCs are detectable due to their association with a wide range of downstream expressions: phenotypic and biochemical—ranging from fitness, antimicrobial susceptibility and virulence [23–27]. In contrast, rrs does not code for a protein per se. Here, NCs are likely to provide a very narrow range of information on the variability in its structure and function [27]. On the other hand, an evaluation of upstream “expression” in rrs due to NCs is more likely to reflect variations in RE activities, as the available multiple action sites are distributed along the length of the rrs [5, 6, 11]. Mutation in rrs of Mycoplasma species at position 997 (C>T) in RE site of AluI (AGˆCT) yielded unknown Amplified rDNA Restriction Analysis profiles [28]. Variations in RE maps in rrs with AluI, HaeIII and MspI were reported and selected to distinguish Pseudomonas isolates [29, 30]. NCs as markers are gaining importance as tools for molecular genetic analysis [31], strain discrimination [32] and are being used to gain insights into their evolutionary history [33] and as important prognostic markers [34].

A summary based on all the NC and RE digestion patterns of rrs of C. botulinum strains permit us to propose that ATCC 3502, ATCC 19397/Hall, and Langeland are the closest to each other and were closely related to Loch Maree and Okra, prior to their divergence. High nt identity among ATCC3502, ATCC 19397 and Hall belonging to BoNT/A1 has in fact illustrated their clonal nature [35]. On the other hand, ATCC 3502 seems to have diverged in a different direction, since it had NCs at positions quite different from those observed in ATCC 19397/Hall, and Langeland (Fig. 2). In the study carried out to establish evolutionary relationships among C. botulinum A1 genomes, ATCC19379 has been shown to fall close too Hall A and ATCC3502 [8]. Independent evolution of Eklund 17B appears to have happened after its common origin with Alaska E43, which is supported by the only case of transversion C>A at similar position i.e., 983 in only one copy of rrs in both of them (Table S8). The strains 657 and Kyoto might have originated from a common ancestor, with divergence occurring due to genetic changes in Kyoto. Thus, in spite of their reported diverse geographical origins, these closely related strains can be identified on the basis of the unique in silico RE maps of their rrs and cladistic analysis (Fig. 1). In fact, Pseudomonas spp. originating from diverse biogeographical locations were also found to be genetically quite similar [36].

The evolutionary significance of NCs can be viewed to impart resistance to attacks by REs in these C. botulinum strains. Simultaneous evolution of novel and additional RE sites in 230613, Alaska E43, ATCC 19397/HALL, ATCC 3502, H04402065, Kyoto, Langeland and Loch Maree can be interpreted to have higher susceptibility to RE attack in these organisms. In contrast, 657, BKT015925, Eklund 17B and Okra have evolved to become more resistant to RE attack by not allowing the evolution of any novel RE sites and by loosing a large number of existing RE sites (8–65) (Table S9). ATCC 19397/Hall, ATCC 3502 and Langeland seems to have evolved in a unique manner since they lost RE sites with high frequency (36–44),which can optimize the process of evolution of resistance to REs. Strain 657 added 30 RE sites, which made it more susceptible to RE attack. Eklund 17B is a strain, which can be recognized to have become highly resistant to REs attack on account of higher loss in RE sites and no evolution of novel RE sites.

Conclusion

In conclusion, the presence of NCs and multiple RE sites within a single gene provides opportunities to elucidate the sequence of evolutionary events. NC at the same position within multiple copies of rrs belonging to different C. botulinum strains indicated towards a common origin of ATCC 19397 and Langeland and their subsequent diversifications. Secondly, NCs causing specific changes in RE sites (Fig. 2), and unique RE digestion patterns of rrs can be used as molecular markers for distinguishing closely related strains of C. botulinum.