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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

Inhibition of growth by TPEN is rescued by the addition of Zn2+ ions.(A) Susceptibility to Zn2+ starvation of wild type, ΔadcA and Δlmb  mutants grown in THY medium containing increasing concentrations of the chelating agent TPEN. (B) Inhibition of growth by 50 μM TPEN is rescued by the addition of either 50 μM Zn2+ in wild type as well as in ΔadcA and Δlmb  mutants. Error bars represent the standard deviation.
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pone.0152835.g005: Inhibition of growth by TPEN is rescued by the addition of Zn2+ ions.(A) Susceptibility to Zn2+ starvation of wild type, ΔadcA and Δlmb mutants grown in THY medium containing increasing concentrations of the chelating agent TPEN. (B) Inhibition of growth by 50 μM TPEN is rescued by the addition of either 50 μM Zn2+ in wild type as well as in ΔadcA and Δlmb mutants. Error bars represent the standard deviation.

Mentions: Loss of adcA or lmb expression in the two mutants did not significantly impair their growth rates in THY medium with respect to that of the wild type strain (S1 Fig). Furthermore, growth in the presence of increasing concentrations of TPEN, for both ΔadcA and Δlmb mutants, was also comparable with that of the wild type strain at concentrations up to 25 μM TPEN. Conversely, at 30 μM TPEN only the wild type strain was able to grow and at 35μM TPEN growth was completely inhibited for all strains (Fig 5A). Addition of equimolar amounts of Zn2+ ions to the zinc-depleted medium could rescue growth of both ΔadcA and Δlmb mutant strains (Fig 5B), thus confirming that in S. pyogenes these two SBPs have overlapping roles in zinc acquisition, as already described in S. pneumoniae [8].


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

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

Inhibition of growth by TPEN is rescued by the addition of Zn2+ ions.(A) Susceptibility to Zn2+ starvation of wild type, ΔadcA and Δlmb  mutants grown in THY medium containing increasing concentrations of the chelating agent TPEN. (B) Inhibition of growth by 50 μM TPEN is rescued by the addition of either 50 μM Zn2+ in wild type as well as in ΔadcA and Δlmb  mutants. Error bars represent the standard deviation.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0152835.g005: Inhibition of growth by TPEN is rescued by the addition of Zn2+ ions.(A) Susceptibility to Zn2+ starvation of wild type, ΔadcA and Δlmb mutants grown in THY medium containing increasing concentrations of the chelating agent TPEN. (B) Inhibition of growth by 50 μM TPEN is rescued by the addition of either 50 μM Zn2+ in wild type as well as in ΔadcA and Δlmb mutants. Error bars represent the standard deviation.
Mentions: Loss of adcA or lmb expression in the two mutants did not significantly impair their growth rates in THY medium with respect to that of the wild type strain (S1 Fig). Furthermore, growth in the presence of increasing concentrations of TPEN, for both ΔadcA and Δlmb mutants, was also comparable with that of the wild type strain at concentrations up to 25 μM TPEN. Conversely, at 30 μM TPEN only the wild type strain was able to grow and at 35μM TPEN growth was completely inhibited for all strains (Fig 5A). Addition of equimolar amounts of Zn2+ ions to the zinc-depleted medium could rescue growth of both ΔadcA and Δlmb mutant strains (Fig 5B), thus confirming that in S. pyogenes these two SBPs have overlapping roles in zinc acquisition, as already described in S. pneumoniae [8].

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