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Genomic and physiological variability within Group II (non-proteolytic) Clostridium botulinum.

Stringer SC, Carter AT, Webb MD, Wachnicka E, Crossman LC, Sebaihia M, Peck MW - BMC Genomics (2013)

Bottom Line: These results were compared with characteristics determined from physiological tests.However, these two subsets did not differ strongly in minimum growth temperature or maximum NaCl concentration for growth.No relationship was found between tellurite resistance and toxin type despite all the tested type B and type F strains carrying tehB, while the sequence was absent or diverged in all type E strains.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Food Research (IFR), Norwich Research Park, Colney, Norwich NR4 7UA, UK. sandra.stringer@ifr.ac.uk

ABSTRACT

Background: Clostridium botulinum is a group of four physiologically and phylogenetically distinct bacteria that produce botulinum neurotoxin. While studies have characterised variability between strains of Group I (proteolytic) C. botulinum, the genetic and physiological variability and relationships between strains within Group II (non-proteolytic) C. botulinum are not well understood. In this study the genome of Group II strain C. botulinum Eklund 17B (NRP) was sequenced and used to construct a whole genome DNA microarray. This was used in a comparative genomic indexing study to compare the relatedness of 43 strains of Group II C. botulinum (14 type B, 24 type E and 5 type F). These results were compared with characteristics determined from physiological tests.

Results: Whole genome indexing showed that strains of Group II C. botulinum isolated from a wide variety of environments over more than 75 years clustered together indicating the genetic background of Group II C. botulinum is stable. Further analysis showed that strains forming type B or type F toxin are closely related with only toxin cluster genes targets being unique to either type. Strains producing type E toxin formed a separate subset. Carbohydrate fermentation tests supported the observation that type B and F strains form a separate subset to type E strains. All the type F strains and most of type B strains produced acid from amylopectin, amylose and glycogen whereas type E strains did not. However, these two subsets did not differ strongly in minimum growth temperature or maximum NaCl concentration for growth. No relationship was found between tellurite resistance and toxin type despite all the tested type B and type F strains carrying tehB, while the sequence was absent or diverged in all type E strains.

Conclusions: Although Group II C. botulinum form a tight genetic group, genomic and physiological analysis indicates there are two distinct subsets within this group. All type B strains and type F strains are in one subset and all type E strains in the other.

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Genomic indexing of 43 strains of Group II C. botulinum. An average linkage hierarchical clustering dendrogram of the C. botulinum strains was created in GeneSpring version 7.0 from microarray data using the Pearson coefficient correlation. Each row of the heatmap represents one strain and consists of a series of vertical bars which represent the CDS content of C. botulinum Eklund 17B (NRP). From left to right these bars are the chromosome from CB17B_0001 to CB17B_3470 then the plasmid from CB17B_P01 to CB17B_P54. The colour of each bar indicates the ratio of the signal from the test strain over that of the index strain. Where the signal from DNA binding is equal for both the test and control strains the bar is coloured yellow and where there is lower binding by the test strain the bar is blue. Where there is no probe the bar is white. The hierarchical clustering dendrogram has been coloured according to neurotoxin type: red =type B, blue =type E and green = type F. Strains were divided into three clusters using a distance of 0.5. Strain Eklund 2B appears slightly separate from the other Cluster 1 strains but this was a result of the culture used on the array lacking the 54 plasmid CDSs. Using only the chromosomal probes, Eklund 2B clustered with the other three strains in Clade 1.
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Figure 5: Genomic indexing of 43 strains of Group II C. botulinum. An average linkage hierarchical clustering dendrogram of the C. botulinum strains was created in GeneSpring version 7.0 from microarray data using the Pearson coefficient correlation. Each row of the heatmap represents one strain and consists of a series of vertical bars which represent the CDS content of C. botulinum Eklund 17B (NRP). From left to right these bars are the chromosome from CB17B_0001 to CB17B_3470 then the plasmid from CB17B_P01 to CB17B_P54. The colour of each bar indicates the ratio of the signal from the test strain over that of the index strain. Where the signal from DNA binding is equal for both the test and control strains the bar is coloured yellow and where there is lower binding by the test strain the bar is blue. Where there is no probe the bar is white. The hierarchical clustering dendrogram has been coloured according to neurotoxin type: red =type B, blue =type E and green = type F. Strains were divided into three clusters using a distance of 0.5. Strain Eklund 2B appears slightly separate from the other Cluster 1 strains but this was a result of the culture used on the array lacking the 54 plasmid CDSs. Using only the chromosomal probes, Eklund 2B clustered with the other three strains in Clade 1.

Mentions: The DNA microarray used for competitive genomic indexing in this study comprised 4160 probes. Each was sixty nucleotides long and tested by BLAST analysis to bind to unique sites in the Group II C. botulinum Eklund 17B (NRP) chromosome and plasmid sequences. These probes represented 3384 chromosomal CDSs and 50 plasmid CDSs which is 97.4% of the 3524 total CDSs that were predicted from the sequence data available at that time. This microarray was used to index the CDS content of 43 strains of Group II C. botulinum in relation to the control Eklund 17B (NRP) strain. The hybridization intensity ratio of genomic DNA from the test strain hybridized against Eklund 17B (NRP) DNA for each CDS in chromosome order is shown in Figure 5. The number of CDSs where the test probe signal was within 8-fold of the control signal is shown in Table 2.


Genomic and physiological variability within Group II (non-proteolytic) Clostridium botulinum.

Stringer SC, Carter AT, Webb MD, Wachnicka E, Crossman LC, Sebaihia M, Peck MW - BMC Genomics (2013)

Genomic indexing of 43 strains of Group II C. botulinum. An average linkage hierarchical clustering dendrogram of the C. botulinum strains was created in GeneSpring version 7.0 from microarray data using the Pearson coefficient correlation. Each row of the heatmap represents one strain and consists of a series of vertical bars which represent the CDS content of C. botulinum Eklund 17B (NRP). From left to right these bars are the chromosome from CB17B_0001 to CB17B_3470 then the plasmid from CB17B_P01 to CB17B_P54. The colour of each bar indicates the ratio of the signal from the test strain over that of the index strain. Where the signal from DNA binding is equal for both the test and control strains the bar is coloured yellow and where there is lower binding by the test strain the bar is blue. Where there is no probe the bar is white. The hierarchical clustering dendrogram has been coloured according to neurotoxin type: red =type B, blue =type E and green = type F. Strains were divided into three clusters using a distance of 0.5. Strain Eklund 2B appears slightly separate from the other Cluster 1 strains but this was a result of the culture used on the array lacking the 54 plasmid CDSs. Using only the chromosomal probes, Eklund 2B clustered with the other three strains in Clade 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC3672017&req=5

Figure 5: Genomic indexing of 43 strains of Group II C. botulinum. An average linkage hierarchical clustering dendrogram of the C. botulinum strains was created in GeneSpring version 7.0 from microarray data using the Pearson coefficient correlation. Each row of the heatmap represents one strain and consists of a series of vertical bars which represent the CDS content of C. botulinum Eklund 17B (NRP). From left to right these bars are the chromosome from CB17B_0001 to CB17B_3470 then the plasmid from CB17B_P01 to CB17B_P54. The colour of each bar indicates the ratio of the signal from the test strain over that of the index strain. Where the signal from DNA binding is equal for both the test and control strains the bar is coloured yellow and where there is lower binding by the test strain the bar is blue. Where there is no probe the bar is white. The hierarchical clustering dendrogram has been coloured according to neurotoxin type: red =type B, blue =type E and green = type F. Strains were divided into three clusters using a distance of 0.5. Strain Eklund 2B appears slightly separate from the other Cluster 1 strains but this was a result of the culture used on the array lacking the 54 plasmid CDSs. Using only the chromosomal probes, Eklund 2B clustered with the other three strains in Clade 1.
Mentions: The DNA microarray used for competitive genomic indexing in this study comprised 4160 probes. Each was sixty nucleotides long and tested by BLAST analysis to bind to unique sites in the Group II C. botulinum Eklund 17B (NRP) chromosome and plasmid sequences. These probes represented 3384 chromosomal CDSs and 50 plasmid CDSs which is 97.4% of the 3524 total CDSs that were predicted from the sequence data available at that time. This microarray was used to index the CDS content of 43 strains of Group II C. botulinum in relation to the control Eklund 17B (NRP) strain. The hybridization intensity ratio of genomic DNA from the test strain hybridized against Eklund 17B (NRP) DNA for each CDS in chromosome order is shown in Figure 5. The number of CDSs where the test probe signal was within 8-fold of the control signal is shown in Table 2.

Bottom Line: These results were compared with characteristics determined from physiological tests.However, these two subsets did not differ strongly in minimum growth temperature or maximum NaCl concentration for growth.No relationship was found between tellurite resistance and toxin type despite all the tested type B and type F strains carrying tehB, while the sequence was absent or diverged in all type E strains.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Food Research (IFR), Norwich Research Park, Colney, Norwich NR4 7UA, UK. sandra.stringer@ifr.ac.uk

ABSTRACT

Background: Clostridium botulinum is a group of four physiologically and phylogenetically distinct bacteria that produce botulinum neurotoxin. While studies have characterised variability between strains of Group I (proteolytic) C. botulinum, the genetic and physiological variability and relationships between strains within Group II (non-proteolytic) C. botulinum are not well understood. In this study the genome of Group II strain C. botulinum Eklund 17B (NRP) was sequenced and used to construct a whole genome DNA microarray. This was used in a comparative genomic indexing study to compare the relatedness of 43 strains of Group II C. botulinum (14 type B, 24 type E and 5 type F). These results were compared with characteristics determined from physiological tests.

Results: Whole genome indexing showed that strains of Group II C. botulinum isolated from a wide variety of environments over more than 75 years clustered together indicating the genetic background of Group II C. botulinum is stable. Further analysis showed that strains forming type B or type F toxin are closely related with only toxin cluster genes targets being unique to either type. Strains producing type E toxin formed a separate subset. Carbohydrate fermentation tests supported the observation that type B and F strains form a separate subset to type E strains. All the type F strains and most of type B strains produced acid from amylopectin, amylose and glycogen whereas type E strains did not. However, these two subsets did not differ strongly in minimum growth temperature or maximum NaCl concentration for growth. No relationship was found between tellurite resistance and toxin type despite all the tested type B and type F strains carrying tehB, while the sequence was absent or diverged in all type E strains.

Conclusions: Although Group II C. botulinum form a tight genetic group, genomic and physiological analysis indicates there are two distinct subsets within this group. All type B strains and type F strains are in one subset and all type E strains in the other.

Show MeSH
Related in: MedlinePlus