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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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Susceptibility to Zn2+ starvation.Susceptibility to Zn2+ starvation of wild type, ΔadcA  mutant and complemented strains grown in THY medium containing increasing concentrations of the chelating agent N,N,N’N’-Tetrakis (2-pyridylmethyl)-1,2-ethylenediamine (TPEN). Error bars represent the standard deviation.
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pone.0152835.g002: Susceptibility to Zn2+ starvation.Susceptibility to Zn2+ starvation of wild type, ΔadcA mutant and complemented strains grown in THY medium containing increasing concentrations of the chelating agent N,N,N’N’-Tetrakis (2-pyridylmethyl)-1,2-ethylenediamine (TPEN). Error bars represent the standard deviation.

Mentions: Since the gene product of adcA in other streptococci is known to be involved primarily in zinc uptake and homeostasis, we analysed the sensitivity to zinc starvation of the ∆adcA mutant and compared it with that of the wild type strain. The same mutant complemented with the pMU1328-adcA construct or carrying the empty pMU1328 vector were also included in our analysis as controls (Fig 2). The chelating agent N,N,N’,N’-Tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), a cell permeable high-affinity Zn2+ chelator used to reduce intra- and extra-cellular concentration of zinc in zinc homeostasis studies [32][16], was added at increasing concentrations to the growth medium of the ∆adcA mutant and MGAS5005 wild type strain. The ΔadcA mutant showed a higher susceptibility to zinc starvation. Growth was nearly abolished in the mutant by the addition of 30 µM TPEN, while it was only slightly decreased in the wild type or in the complemented strain (Fig 2). Complete inhibition of growth was observed at 35 μM TPEN for all strains, except for the ΔadcA mutant complemented with the pMU1328-adcA construct which showed some residual growth at 35 µM TPEN, possibly due to the higher level of AdcA expression in this strain (Fig 1C).


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

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

Susceptibility to Zn2+ starvation.Susceptibility to Zn2+ starvation of wild type, ΔadcA  mutant and complemented strains grown in THY medium containing increasing concentrations of the chelating agent N,N,N’N’-Tetrakis (2-pyridylmethyl)-1,2-ethylenediamine (TPEN). Error bars represent the standard deviation.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0152835.g002: Susceptibility to Zn2+ starvation.Susceptibility to Zn2+ starvation of wild type, ΔadcA mutant and complemented strains grown in THY medium containing increasing concentrations of the chelating agent N,N,N’N’-Tetrakis (2-pyridylmethyl)-1,2-ethylenediamine (TPEN). Error bars represent the standard deviation.
Mentions: Since the gene product of adcA in other streptococci is known to be involved primarily in zinc uptake and homeostasis, we analysed the sensitivity to zinc starvation of the ∆adcA mutant and compared it with that of the wild type strain. The same mutant complemented with the pMU1328-adcA construct or carrying the empty pMU1328 vector were also included in our analysis as controls (Fig 2). The chelating agent N,N,N’,N’-Tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), a cell permeable high-affinity Zn2+ chelator used to reduce intra- and extra-cellular concentration of zinc in zinc homeostasis studies [32][16], was added at increasing concentrations to the growth medium of the ∆adcA mutant and MGAS5005 wild type strain. The ΔadcA mutant showed a higher susceptibility to zinc starvation. Growth was nearly abolished in the mutant by the addition of 30 µM TPEN, while it was only slightly decreased in the wild type or in the complemented strain (Fig 2). Complete inhibition of growth was observed at 35 μM TPEN for all strains, except for the ΔadcA mutant complemented with the pMU1328-adcA construct which showed some residual growth at 35 µM TPEN, possibly due to the higher level of AdcA expression in this strain (Fig 1C).

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