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A novel streptococcal integrative conjugative element involved in iron acquisition.

Heather Z, Holden MT, Steward KF, Parkhill J, Song L, Challis GL, Robinson C, Davis-Poynter N, Waller AS - Mol. Microbiol. (2008)

Bottom Line: Deletion of eqbA resulted in a small-colony phenotype.Quantification of (55)Fe accumulation and sensitivity to streptonigrin suggested that equibactin is secreted by S. equi and that the eqbH, eqbI and eqbJ genes are required for its associated iron import.In agreement with a structure-based model of equibactin synthesis, supplementation of chemically defined media with salicylate was required for equibactin production.

View Article: PubMed Central - PubMed

Affiliation: Centre for Preventive Medicine, Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk, UK.

ABSTRACT
In this study, we determined the function of a novel non-ribosomal peptide synthetase (NRPS) system carried by a streptococcal integrative conjugative element (ICE), ICESe2. The NRPS shares similarity with the yersiniabactin system found in the high-pathogenicity island of Yersinia sp. and is the first of its kind to be identified in streptococci. We named the NRPS product 'equibactin' and genes of this locus eqbA-N. ICESe2, although absolutely conserved in Streptococcus equi, the causative agent of equine strangles, was absent from all strains of the closely related opportunistic pathogen Streptococcus zooepidemicus. Binding of EqbA, a DtxR-like regulator, to the eqbB promoter was increased in the presence of cations. Deletion of eqbA resulted in a small-colony phenotype. Further deletion of the irp2 homologue eqbE, or the genes eqbH, eqbI and eqbJ encoding a putative ABC transporter, or addition of the iron chelator nitrilotriacetate, reversed this phenotype, implicating iron toxicity. Quantification of (55)Fe accumulation and sensitivity to streptonigrin suggested that equibactin is secreted by S. equi and that the eqbH, eqbI and eqbJ genes are required for its associated iron import. In agreement with a structure-based model of equibactin synthesis, supplementation of chemically defined media with salicylate was required for equibactin production.

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Phenotypic effects of deletions in the eqb gene cluster. A. Photographs show colonies of wild-type, ΔeqbA, ΔeqbAE, ΔeqbHIJA, ΔeqbKLA and ΔeqbAΔftsB S. equi strains grown overnight on THA and the increase in colony size of the ΔeqbA, ΔeqbKLA and ΔeqbAΔftsB strains grown on THA supplemented with 2 mM NTA. B. Fold increase in eqbE transcript level in the ΔeqbA strain relative to the wild-type 4047 strain, which has been normalized to one (mean ± standard error mean, n = 3). C. Quantification of 55Fe accumulation by different S. equi strains (mean ± standard error mean, n = 3). The difference in 55Fe accumulation between the ΔeqbA strain and the ΔeqbAE, ΔeqbHIJA and wild-type strains was found to be statistically significant using two-sample Wilcoxon rank-sum (Mann–Whitney) tests (P = 0.05, n = 3). D. Quantification of 55Fe accumulation by S. equi strain ΔeqbAE cross-fed with filter-sterilized culture supernatant from different S. equi strains grown to stationary phase.
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fig03: Phenotypic effects of deletions in the eqb gene cluster. A. Photographs show colonies of wild-type, ΔeqbA, ΔeqbAE, ΔeqbHIJA, ΔeqbKLA and ΔeqbAΔftsB S. equi strains grown overnight on THA and the increase in colony size of the ΔeqbA, ΔeqbKLA and ΔeqbAΔftsB strains grown on THA supplemented with 2 mM NTA. B. Fold increase in eqbE transcript level in the ΔeqbA strain relative to the wild-type 4047 strain, which has been normalized to one (mean ± standard error mean, n = 3). C. Quantification of 55Fe accumulation by different S. equi strains (mean ± standard error mean, n = 3). The difference in 55Fe accumulation between the ΔeqbA strain and the ΔeqbAE, ΔeqbHIJA and wild-type strains was found to be statistically significant using two-sample Wilcoxon rank-sum (Mann–Whitney) tests (P = 0.05, n = 3). D. Quantification of 55Fe accumulation by S. equi strain ΔeqbAE cross-fed with filter-sterilized culture supernatant from different S. equi strains grown to stationary phase.

Mentions: To determine the role of EqbA on the regulation of the eqb NRPS, we generated a series of allelic replacement mutants in S. equi strain 4047. Deletion of eqbE had no effect on the growth rate of S. equi on Todd–Hewitt agar (THA). However, deletion of eqbA produced very small colonies (Fig. 3A). We hypothesized that this phenotype resulted from iron toxicity resulting from over-production of the product(s) of the eqb cluster. Over-expression of eqbE was confirmed since deletion of eqbA resulted in a 13-fold increase in eqbE transcript levels (Fig. 3B). The generation of an ΔeqbA, ΔeqbE double deletion strain (ΔeqbAE), which had a large colony phenotype on THA, established that the slow growth of the ΔeqbA strain was as a direct consequence of the function of the eqbE gene product (Fig. 3A). Transformation of the ΔeqbA or ΔeqbAE strains with the pGhost9 plasmid containing a second copy of eqbA under the control of the eqbA promoter or a second copy of eqbE under the control of the eqbB promoter complemented the ΔeqbA and ΔeqbE and induced a large or small colony phenotype respectively (Fig. S2). The lack of an effect on the colony phenotype of S. equi grown in vitro following deletion of eqbE may be due to continued import of cations through the activity of several alternative cation transport systems, which include an HtsABC haem-binding system (SEQ0445 to SEQ0448) (Nygaard et al., 2006), a putative MtsABC Mn2+ and Fe3+ metal transport system (SEQ1658 to SEQ1660) with 80–91% aa sequence identity to that of S. pyogenes (Janulczyk et al., 1999) and a putative FtsABCD Fe3+ ferrichrome transport system (SEQ1836 to SEQ1839) with 59–77% aa sequence identity to that of S. pyogenes (Hanks et al., 2005) (http://www.sanger.ac.uk).


A novel streptococcal integrative conjugative element involved in iron acquisition.

Heather Z, Holden MT, Steward KF, Parkhill J, Song L, Challis GL, Robinson C, Davis-Poynter N, Waller AS - Mol. Microbiol. (2008)

Phenotypic effects of deletions in the eqb gene cluster. A. Photographs show colonies of wild-type, ΔeqbA, ΔeqbAE, ΔeqbHIJA, ΔeqbKLA and ΔeqbAΔftsB S. equi strains grown overnight on THA and the increase in colony size of the ΔeqbA, ΔeqbKLA and ΔeqbAΔftsB strains grown on THA supplemented with 2 mM NTA. B. Fold increase in eqbE transcript level in the ΔeqbA strain relative to the wild-type 4047 strain, which has been normalized to one (mean ± standard error mean, n = 3). C. Quantification of 55Fe accumulation by different S. equi strains (mean ± standard error mean, n = 3). The difference in 55Fe accumulation between the ΔeqbA strain and the ΔeqbAE, ΔeqbHIJA and wild-type strains was found to be statistically significant using two-sample Wilcoxon rank-sum (Mann–Whitney) tests (P = 0.05, n = 3). D. Quantification of 55Fe accumulation by S. equi strain ΔeqbAE cross-fed with filter-sterilized culture supernatant from different S. equi strains grown to stationary phase.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672683&req=5

fig03: Phenotypic effects of deletions in the eqb gene cluster. A. Photographs show colonies of wild-type, ΔeqbA, ΔeqbAE, ΔeqbHIJA, ΔeqbKLA and ΔeqbAΔftsB S. equi strains grown overnight on THA and the increase in colony size of the ΔeqbA, ΔeqbKLA and ΔeqbAΔftsB strains grown on THA supplemented with 2 mM NTA. B. Fold increase in eqbE transcript level in the ΔeqbA strain relative to the wild-type 4047 strain, which has been normalized to one (mean ± standard error mean, n = 3). C. Quantification of 55Fe accumulation by different S. equi strains (mean ± standard error mean, n = 3). The difference in 55Fe accumulation between the ΔeqbA strain and the ΔeqbAE, ΔeqbHIJA and wild-type strains was found to be statistically significant using two-sample Wilcoxon rank-sum (Mann–Whitney) tests (P = 0.05, n = 3). D. Quantification of 55Fe accumulation by S. equi strain ΔeqbAE cross-fed with filter-sterilized culture supernatant from different S. equi strains grown to stationary phase.
Mentions: To determine the role of EqbA on the regulation of the eqb NRPS, we generated a series of allelic replacement mutants in S. equi strain 4047. Deletion of eqbE had no effect on the growth rate of S. equi on Todd–Hewitt agar (THA). However, deletion of eqbA produced very small colonies (Fig. 3A). We hypothesized that this phenotype resulted from iron toxicity resulting from over-production of the product(s) of the eqb cluster. Over-expression of eqbE was confirmed since deletion of eqbA resulted in a 13-fold increase in eqbE transcript levels (Fig. 3B). The generation of an ΔeqbA, ΔeqbE double deletion strain (ΔeqbAE), which had a large colony phenotype on THA, established that the slow growth of the ΔeqbA strain was as a direct consequence of the function of the eqbE gene product (Fig. 3A). Transformation of the ΔeqbA or ΔeqbAE strains with the pGhost9 plasmid containing a second copy of eqbA under the control of the eqbA promoter or a second copy of eqbE under the control of the eqbB promoter complemented the ΔeqbA and ΔeqbE and induced a large or small colony phenotype respectively (Fig. S2). The lack of an effect on the colony phenotype of S. equi grown in vitro following deletion of eqbE may be due to continued import of cations through the activity of several alternative cation transport systems, which include an HtsABC haem-binding system (SEQ0445 to SEQ0448) (Nygaard et al., 2006), a putative MtsABC Mn2+ and Fe3+ metal transport system (SEQ1658 to SEQ1660) with 80–91% aa sequence identity to that of S. pyogenes (Janulczyk et al., 1999) and a putative FtsABCD Fe3+ ferrichrome transport system (SEQ1836 to SEQ1839) with 59–77% aa sequence identity to that of S. pyogenes (Hanks et al., 2005) (http://www.sanger.ac.uk).

Bottom Line: Deletion of eqbA resulted in a small-colony phenotype.Quantification of (55)Fe accumulation and sensitivity to streptonigrin suggested that equibactin is secreted by S. equi and that the eqbH, eqbI and eqbJ genes are required for its associated iron import.In agreement with a structure-based model of equibactin synthesis, supplementation of chemically defined media with salicylate was required for equibactin production.

View Article: PubMed Central - PubMed

Affiliation: Centre for Preventive Medicine, Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk, UK.

ABSTRACT
In this study, we determined the function of a novel non-ribosomal peptide synthetase (NRPS) system carried by a streptococcal integrative conjugative element (ICE), ICESe2. The NRPS shares similarity with the yersiniabactin system found in the high-pathogenicity island of Yersinia sp. and is the first of its kind to be identified in streptococci. We named the NRPS product 'equibactin' and genes of this locus eqbA-N. ICESe2, although absolutely conserved in Streptococcus equi, the causative agent of equine strangles, was absent from all strains of the closely related opportunistic pathogen Streptococcus zooepidemicus. Binding of EqbA, a DtxR-like regulator, to the eqbB promoter was increased in the presence of cations. Deletion of eqbA resulted in a small-colony phenotype. Further deletion of the irp2 homologue eqbE, or the genes eqbH, eqbI and eqbJ encoding a putative ABC transporter, or addition of the iron chelator nitrilotriacetate, reversed this phenotype, implicating iron toxicity. Quantification of (55)Fe accumulation and sensitivity to streptonigrin suggested that equibactin is secreted by S. equi and that the eqbH, eqbI and eqbJ genes are required for its associated iron import. In agreement with a structure-based model of equibactin synthesis, supplementation of chemically defined media with salicylate was required for equibactin production.

Show MeSH
Related in: MedlinePlus