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Zn2+ Uptake in Streptococcus pyogenes: Characterization of adcA and lmb Null Mutants.

Tedde V, Rosini R, Galeotti CL - PLoS ONE (2016)

Bottom Line: An effective regulation of metal ion homeostasis is essential for the growth of microorganisms in any environment and in pathogenic bacteria is strongly associated with their ability to invade and colonise their hosts.In contrast, Lmb and HtpA were expressed at detectable levels only during growth in the presence of low zinc concentrations or in the adcA mutant, when expression of lmb is required to compensate for the lack of adcA expression.In the latter case, Lmb and HtpA were overexpressed by several fold, thus indicating that also in GAS AdcA is a zinc-specific importer and, although it shares this function with Lmb, the two substrate-binding proteins do not show fully overlapping roles in zinc homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Research Centre, GlaxoSmithKline Vaccines S.r.l., Siena, Italy.

ABSTRACT
An effective regulation of metal ion homeostasis is essential for the growth of microorganisms in any environment and in pathogenic bacteria is strongly associated with their ability to invade and colonise their hosts. To gain a better insight into zinc acquisition in Group A Streptococcus (GAS) we characterized deletion mutants of the adcA and lmb genes of Streptococcus pyogenes strain MGAS5005 encoding the orthologues of AdcA and AdcAII, the two surface lipoproteins with partly redundant roles in zinc homeostasis in Streptococcus pneumoniae. Null adcA and lmb mutants were analysed for their capability to grow in zinc-depleted conditions and were found to be more susceptible to zinc starvation, a phenotype that could be rescued by the addition of Zn2+ ions to the growth medium. Expression of AdcA, Lmb and HtpA, the polyhistidine triad protein encoded by the gene adjacent to lmb, during growth under conditions of limited zinc availability was examined by Western blot analysis in wild type and mutant strains. In the wild type strain, AdcA was always present with little variation in expression levels between conditions of excess or limited zinc availability. In contrast, Lmb and HtpA were expressed at detectable levels only during growth in the presence of low zinc concentrations or in the adcA mutant, when expression of lmb is required to compensate for the lack of adcA expression. In the latter case, Lmb and HtpA were overexpressed by several fold, thus indicating that also in GAS AdcA is a zinc-specific importer and, although it shares this function with Lmb, the two substrate-binding proteins do not show fully overlapping roles in zinc homeostasis.

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Related in: MedlinePlus

Construction of Δlmb  mutants.(A) Schematic representation of the lmb locus in S. pyogenes MGAS5005 wild type and Δlmb deletion mutants. (B) Western blot analysis of Lmb expression using anti-Lmb specific antibodies in total cell extracts of wild type and of a Δlmb  mutant grown in THY medium or in THY with the addition of 35 μM TPEN and 15 μM ZnCl2. The latter growth medium was tested since it was observed to give higher expression of Lmb in our experimental conditions.
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pone.0152835.g004: Construction of Δlmb mutants.(A) Schematic representation of the lmb locus in S. pyogenes MGAS5005 wild type and Δlmb deletion mutants. (B) Western blot analysis of Lmb expression using anti-Lmb specific antibodies in total cell extracts of wild type and of a Δlmb mutant grown in THY medium or in THY with the addition of 35 μM TPEN and 15 μM ZnCl2. The latter growth medium was tested since it was observed to give higher expression of Lmb in our experimental conditions.

Mentions: An in-frame deletion mutant of lmb/lsp has already been described in the S. pyogenes strain HSC5 [36] and shown to have a role in zinc homeostasis and pathogenesis [16]. However, due to the variability in genetic composition between different S. pyogenes strains [37] and the complexity of zinc homeostasis control in any biological system, we generated a Δlmb mutant in strain MGAS5005 in order to investigate the contribution of Lmb to zinc uptake in the same genetic background used for characterizing AdcA. The map of the locus in the lmb mutant is outlined in Fig 4A and the Western blot analysis carried out to confirm the absence of Lmb expression in the mutant strain is given in Fig 4B.


Zn2+ Uptake in Streptococcus pyogenes: Characterization of adcA and lmb Null Mutants.

Tedde V, Rosini R, Galeotti CL - PLoS ONE (2016)

Construction of Δlmb  mutants.(A) Schematic representation of the lmb locus in S. pyogenes MGAS5005 wild type and Δlmb deletion mutants. (B) Western blot analysis of Lmb expression using anti-Lmb specific antibodies in total cell extracts of wild type and of a Δlmb  mutant grown in THY medium or in THY with the addition of 35 μM TPEN and 15 μM ZnCl2. The latter growth medium was tested since it was observed to give higher expression of Lmb in our experimental conditions.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4816340&req=5

pone.0152835.g004: Construction of Δlmb mutants.(A) Schematic representation of the lmb locus in S. pyogenes MGAS5005 wild type and Δlmb deletion mutants. (B) Western blot analysis of Lmb expression using anti-Lmb specific antibodies in total cell extracts of wild type and of a Δlmb mutant grown in THY medium or in THY with the addition of 35 μM TPEN and 15 μM ZnCl2. The latter growth medium was tested since it was observed to give higher expression of Lmb in our experimental conditions.
Mentions: An in-frame deletion mutant of lmb/lsp has already been described in the S. pyogenes strain HSC5 [36] and shown to have a role in zinc homeostasis and pathogenesis [16]. However, due to the variability in genetic composition between different S. pyogenes strains [37] and the complexity of zinc homeostasis control in any biological system, we generated a Δlmb mutant in strain MGAS5005 in order to investigate the contribution of Lmb to zinc uptake in the same genetic background used for characterizing AdcA. The map of the locus in the lmb mutant is outlined in Fig 4A and the Western blot analysis carried out to confirm the absence of Lmb expression in the mutant strain is given in Fig 4B.

Bottom Line: An effective regulation of metal ion homeostasis is essential for the growth of microorganisms in any environment and in pathogenic bacteria is strongly associated with their ability to invade and colonise their hosts.In contrast, Lmb and HtpA were expressed at detectable levels only during growth in the presence of low zinc concentrations or in the adcA mutant, when expression of lmb is required to compensate for the lack of adcA expression.In the latter case, Lmb and HtpA were overexpressed by several fold, thus indicating that also in GAS AdcA is a zinc-specific importer and, although it shares this function with Lmb, the two substrate-binding proteins do not show fully overlapping roles in zinc homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Research Centre, GlaxoSmithKline Vaccines S.r.l., Siena, Italy.

ABSTRACT
An effective regulation of metal ion homeostasis is essential for the growth of microorganisms in any environment and in pathogenic bacteria is strongly associated with their ability to invade and colonise their hosts. To gain a better insight into zinc acquisition in Group A Streptococcus (GAS) we characterized deletion mutants of the adcA and lmb genes of Streptococcus pyogenes strain MGAS5005 encoding the orthologues of AdcA and AdcAII, the two surface lipoproteins with partly redundant roles in zinc homeostasis in Streptococcus pneumoniae. Null adcA and lmb mutants were analysed for their capability to grow in zinc-depleted conditions and were found to be more susceptible to zinc starvation, a phenotype that could be rescued by the addition of Zn2+ ions to the growth medium. Expression of AdcA, Lmb and HtpA, the polyhistidine triad protein encoded by the gene adjacent to lmb, during growth under conditions of limited zinc availability was examined by Western blot analysis in wild type and mutant strains. In the wild type strain, AdcA was always present with little variation in expression levels between conditions of excess or limited zinc availability. In contrast, Lmb and HtpA were expressed at detectable levels only during growth in the presence of low zinc concentrations or in the adcA mutant, when expression of lmb is required to compensate for the lack of adcA expression. In the latter case, Lmb and HtpA were overexpressed by several fold, thus indicating that also in GAS AdcA is a zinc-specific importer and, although it shares this function with Lmb, the two substrate-binding proteins do not show fully overlapping roles in zinc homeostasis.

Show MeSH
Related in: MedlinePlus