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Investigations into genome diversity of Haemophilus influenzae using whole genome sequencing of clinical isolates and laboratory transformants.

Power PM, Bentley SD, Parkhill J, Moxon ER, Hood DW - BMC Microbiol. (2012)

Bottom Line: Haemophilus influenzae is an important human commensal pathogen associated with significant levels of disease.When compared by Mauve alignment these indicated groupings of H. influenzae that were consistent with previously published analyses; capsule expressing strains fell into two distinct groups and those of serotype b (Hib) were found in two closely positioned lineages.For 18 Hib strains representing both lineages we found many discrete regions (up to 40% of the total genome) displaying sequence variation when compared to a common reference strain.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Oxford Department of Paediatrics, Medical Sciences Division, John Radcliffe Hospital, Headington, Oxford, UK. .

ABSTRACT

Background: Haemophilus influenzae is an important human commensal pathogen associated with significant levels of disease. High-throughput DNA sequencing was used to investigate differences in genome content within this species.

Results: Genomic DNA sequence was obtained from 85 strains of H. influenzae and from other related species, selected based on geographical site of isolation, disease association and documented genotypic and phenotypic differences. When compared by Mauve alignment these indicated groupings of H. influenzae that were consistent with previously published analyses; capsule expressing strains fell into two distinct groups and those of serotype b (Hib) were found in two closely positioned lineages. For 18 Hib strains representing both lineages we found many discrete regions (up to 40% of the total genome) displaying sequence variation when compared to a common reference strain. Evidence that this naturally occurring pattern of inter-strain variation in H. influenzae can be mediated by transformation was obtained through sequencing DNA obtained from a pool of 200 independent transformants of a recipient (strain Rd) using donor DNA from a heterologous Hib strain (Eagan).

Conclusion: Much of the inter-strain variation in genome sequence in H. influenzae is likely the result of inter-strain exchanges of DNA, most plausibly through transformation.

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Related in: MedlinePlus

Frequency of EaganstrRand Eagan SNPs in the Rd+EaganstrRand Rd+Eagan transformants. Panel A; Location and frequency of EaganstrR specific SNPs plotted as estimated number of strains (y-axis) against location in RdKW20 genome sequence (x-axis) using SNPSeeker. MAQ was used to identify SNPs in the pooled sequences from 200 transformants. The location of the strR point mutation is indicated. Panel B; A magnified view of one region marked on Panel A showing a putative secondary transformation event. The extent of the chromosomal region involved with this predicted transformation event (13 kbp) is marked. Panel C; A magnified view of the primary transformation event from Panel A with the location of the strR point mutation marked. Panel D; The location and frequency of Eagan-specific SNPs in the genome of pooled Rd+Eagan transformants (200); Eagan unmarked (wild-type) genomic DNA was used as the donor.
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Figure 4: Frequency of EaganstrRand Eagan SNPs in the Rd+EaganstrRand Rd+Eagan transformants. Panel A; Location and frequency of EaganstrR specific SNPs plotted as estimated number of strains (y-axis) against location in RdKW20 genome sequence (x-axis) using SNPSeeker. MAQ was used to identify SNPs in the pooled sequences from 200 transformants. The location of the strR point mutation is indicated. Panel B; A magnified view of one region marked on Panel A showing a putative secondary transformation event. The extent of the chromosomal region involved with this predicted transformation event (13 kbp) is marked. Panel C; A magnified view of the primary transformation event from Panel A with the location of the strR point mutation marked. Panel D; The location and frequency of Eagan-specific SNPs in the genome of pooled Rd+Eagan transformants (200); Eagan unmarked (wild-type) genomic DNA was used as the donor.

Mentions: High throughput sequencing provides a useful experimental tool to examine in detail the recombination events associated with the transfer and exchange of DNA between H. influenzae strains through transformation. To this end, we investigated the transformation of DNA from a Hib strain donor into a high efficiency recipient strain. To ensure that each transformant was the result of a recombination event we used a spontaneous, high level streptomycin resistant (strR) derivative of strain Eagan (EaganstrR), possessing a point mutation in rpoB. Spontaneous strR mutants were infrequent (<10-10 in control transformations of Rd using streptomycin-sensitive Eagan DNA). Compared to strain Rd, the donor strain Eagan genome sequence had 18,789 SNPs relatively uniformly distributed throughout the genome (an average density of 10.3 SNPs per kbp) including the region around rpoB, the location of the strR mutation. Following transformation and selection on streptomycin, 200 independent Rd+EaganstrR colonies were pooled, the genomic DNA sequenced and mapped to the Rd reference genome sequence using the MAQ programme to identify SNPs. The number of Rd+EaganstrR transformants carrying each SNP was estimated from the pooled sequence using the SNPSeeker script[18] and is plotted in Figure‚ÄČ4. 4,501 SNPs consistent with transfer from Eagan (i.e. they were in the same genome location as the Eagan SNPs identified above) were found in the Rd+EaganstrR transformants. We identified 202 SNPs that were common to all respective sequence reads, were not linked closely to other SNPs and were found in both Rd+EaganstrR and Rd+Eagan transformants obtained in control experiments using non-strR Eagan DNA as donor. We conclude that these SNPs were consistent with, and most likely explained by, errors within the reported Rd genome sequence published in 1995. Another possibility, not mutually exclusive with sequencing errors, could be sequence drift in our laboratory strain (RM118) when compared to the sequenced isolate (Rd KW20). This level of error is similar to the several hundred SNPs reported upon re-sequencing of strain Rd by other investigators[17] and comparable with the 243 discrepancies found between the original 1997 E. coli strain MG1655 genome sequence[19] and the 2006 re-sequencing[20] of the same strain.


Investigations into genome diversity of Haemophilus influenzae using whole genome sequencing of clinical isolates and laboratory transformants.

Power PM, Bentley SD, Parkhill J, Moxon ER, Hood DW - BMC Microbiol. (2012)

Frequency of EaganstrRand Eagan SNPs in the Rd+EaganstrRand Rd+Eagan transformants. Panel A; Location and frequency of EaganstrR specific SNPs plotted as estimated number of strains (y-axis) against location in RdKW20 genome sequence (x-axis) using SNPSeeker. MAQ was used to identify SNPs in the pooled sequences from 200 transformants. The location of the strR point mutation is indicated. Panel B; A magnified view of one region marked on Panel A showing a putative secondary transformation event. The extent of the chromosomal region involved with this predicted transformation event (13 kbp) is marked. Panel C; A magnified view of the primary transformation event from Panel A with the location of the strR point mutation marked. Panel D; The location and frequency of Eagan-specific SNPs in the genome of pooled Rd+Eagan transformants (200); Eagan unmarked (wild-type) genomic DNA was used as the donor.
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Figure 4: Frequency of EaganstrRand Eagan SNPs in the Rd+EaganstrRand Rd+Eagan transformants. Panel A; Location and frequency of EaganstrR specific SNPs plotted as estimated number of strains (y-axis) against location in RdKW20 genome sequence (x-axis) using SNPSeeker. MAQ was used to identify SNPs in the pooled sequences from 200 transformants. The location of the strR point mutation is indicated. Panel B; A magnified view of one region marked on Panel A showing a putative secondary transformation event. The extent of the chromosomal region involved with this predicted transformation event (13 kbp) is marked. Panel C; A magnified view of the primary transformation event from Panel A with the location of the strR point mutation marked. Panel D; The location and frequency of Eagan-specific SNPs in the genome of pooled Rd+Eagan transformants (200); Eagan unmarked (wild-type) genomic DNA was used as the donor.
Mentions: High throughput sequencing provides a useful experimental tool to examine in detail the recombination events associated with the transfer and exchange of DNA between H. influenzae strains through transformation. To this end, we investigated the transformation of DNA from a Hib strain donor into a high efficiency recipient strain. To ensure that each transformant was the result of a recombination event we used a spontaneous, high level streptomycin resistant (strR) derivative of strain Eagan (EaganstrR), possessing a point mutation in rpoB. Spontaneous strR mutants were infrequent (<10-10 in control transformations of Rd using streptomycin-sensitive Eagan DNA). Compared to strain Rd, the donor strain Eagan genome sequence had 18,789 SNPs relatively uniformly distributed throughout the genome (an average density of 10.3 SNPs per kbp) including the region around rpoB, the location of the strR mutation. Following transformation and selection on streptomycin, 200 independent Rd+EaganstrR colonies were pooled, the genomic DNA sequenced and mapped to the Rd reference genome sequence using the MAQ programme to identify SNPs. The number of Rd+EaganstrR transformants carrying each SNP was estimated from the pooled sequence using the SNPSeeker script[18] and is plotted in Figure‚ÄČ4. 4,501 SNPs consistent with transfer from Eagan (i.e. they were in the same genome location as the Eagan SNPs identified above) were found in the Rd+EaganstrR transformants. We identified 202 SNPs that were common to all respective sequence reads, were not linked closely to other SNPs and were found in both Rd+EaganstrR and Rd+Eagan transformants obtained in control experiments using non-strR Eagan DNA as donor. We conclude that these SNPs were consistent with, and most likely explained by, errors within the reported Rd genome sequence published in 1995. Another possibility, not mutually exclusive with sequencing errors, could be sequence drift in our laboratory strain (RM118) when compared to the sequenced isolate (Rd KW20). This level of error is similar to the several hundred SNPs reported upon re-sequencing of strain Rd by other investigators[17] and comparable with the 243 discrepancies found between the original 1997 E. coli strain MG1655 genome sequence[19] and the 2006 re-sequencing[20] of the same strain.

Bottom Line: Haemophilus influenzae is an important human commensal pathogen associated with significant levels of disease.When compared by Mauve alignment these indicated groupings of H. influenzae that were consistent with previously published analyses; capsule expressing strains fell into two distinct groups and those of serotype b (Hib) were found in two closely positioned lineages.For 18 Hib strains representing both lineages we found many discrete regions (up to 40% of the total genome) displaying sequence variation when compared to a common reference strain.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Oxford Department of Paediatrics, Medical Sciences Division, John Radcliffe Hospital, Headington, Oxford, UK. .

ABSTRACT

Background: Haemophilus influenzae is an important human commensal pathogen associated with significant levels of disease. High-throughput DNA sequencing was used to investigate differences in genome content within this species.

Results: Genomic DNA sequence was obtained from 85 strains of H. influenzae and from other related species, selected based on geographical site of isolation, disease association and documented genotypic and phenotypic differences. When compared by Mauve alignment these indicated groupings of H. influenzae that were consistent with previously published analyses; capsule expressing strains fell into two distinct groups and those of serotype b (Hib) were found in two closely positioned lineages. For 18 Hib strains representing both lineages we found many discrete regions (up to 40% of the total genome) displaying sequence variation when compared to a common reference strain. Evidence that this naturally occurring pattern of inter-strain variation in H. influenzae can be mediated by transformation was obtained through sequencing DNA obtained from a pool of 200 independent transformants of a recipient (strain Rd) using donor DNA from a heterologous Hib strain (Eagan).

Conclusion: Much of the inter-strain variation in genome sequence in H. influenzae is likely the result of inter-strain exchanges of DNA, most plausibly through transformation.

Show MeSH
Related in: MedlinePlus