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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 adcA  mutants.(A) Schematic representation of the adcA locus in S. pyogenes MGAS5005 wild type and ΔadcA deletion mutants. (B) Map of plasmid pMU1328 used for complementation of the ΔadcA deletion mutants. (C) Western blot analysis of AdcA expression in total cell extracts of wild type, three independent ΔadcA  mutants and a ΔadcA complemented with the pMU1328-adcA construct using anti-AdcA specific antibodies.
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pone.0152835.g001: Construction of adcA mutants.(A) Schematic representation of the adcA locus in S. pyogenes MGAS5005 wild type and ΔadcA deletion mutants. (B) Map of plasmid pMU1328 used for complementation of the ΔadcA deletion mutants. (C) Western blot analysis of AdcA expression in total cell extracts of wild type, three independent ΔadcA mutants and a ΔadcA complemented with the pMU1328-adcA construct using anti-AdcA specific antibodies.

Mentions: The gene product of adcA in the S. pyogenes strain MGAS5005 has been annotated as the orthologue of the high-affinity zinc uptake system protein ZnuA [17], known to be involved in zinc uptake both in Gram-negative [27] [28] and in Gram-positive bacteria [29]. Isogenic mutants of adcA in strain MGAS5005 were generated as described in the Methods section by using the temperature-sensitive vector pJRS233 carrying the two regions flanking the adcA locus fused together to precisely delete the open reading frame assigned to AdcA (Fig 1A). One of the isogenic ΔadcA mutants was then used to obtain a strain complemented in trans by the wild type adcA gene carried on the expression vector pMU1328 [22]. The shuttle vector pMU1328, originally designed to identify promoter sequences in Streptococcus sanguis, S. lactis and S. cremoris [30], has been shown to be stably maintained in several Streptococcal species [31] [22]. Herein, we show that the pMU1328 replicon is suitable for the efficient expression of a homologous protein also in S. pyogenes. As is evident from Fig 1C, expression of AdcA was completely abolished in the ΔadcA mutant and approximately 2-fold higher than in the wild type strain when the same mutant was complemented with the pMU1328-adcA construct.


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

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

Construction of adcA  mutants.(A) Schematic representation of the adcA locus in S. pyogenes MGAS5005 wild type and ΔadcA deletion mutants. (B) Map of plasmid pMU1328 used for complementation of the ΔadcA deletion mutants. (C) Western blot analysis of AdcA expression in total cell extracts of wild type, three independent ΔadcA  mutants and a ΔadcA complemented with the pMU1328-adcA construct using anti-AdcA specific antibodies.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0152835.g001: Construction of adcA mutants.(A) Schematic representation of the adcA locus in S. pyogenes MGAS5005 wild type and ΔadcA deletion mutants. (B) Map of plasmid pMU1328 used for complementation of the ΔadcA deletion mutants. (C) Western blot analysis of AdcA expression in total cell extracts of wild type, three independent ΔadcA mutants and a ΔadcA complemented with the pMU1328-adcA construct using anti-AdcA specific antibodies.
Mentions: The gene product of adcA in the S. pyogenes strain MGAS5005 has been annotated as the orthologue of the high-affinity zinc uptake system protein ZnuA [17], known to be involved in zinc uptake both in Gram-negative [27] [28] and in Gram-positive bacteria [29]. Isogenic mutants of adcA in strain MGAS5005 were generated as described in the Methods section by using the temperature-sensitive vector pJRS233 carrying the two regions flanking the adcA locus fused together to precisely delete the open reading frame assigned to AdcA (Fig 1A). One of the isogenic ΔadcA mutants was then used to obtain a strain complemented in trans by the wild type adcA gene carried on the expression vector pMU1328 [22]. The shuttle vector pMU1328, originally designed to identify promoter sequences in Streptococcus sanguis, S. lactis and S. cremoris [30], has been shown to be stably maintained in several Streptococcal species [31] [22]. Herein, we show that the pMU1328 replicon is suitable for the efficient expression of a homologous protein also in S. pyogenes. As is evident from Fig 1C, expression of AdcA was completely abolished in the ΔadcA mutant and approximately 2-fold higher than in the wild type strain when the same mutant was complemented with the pMU1328-adcA construct.

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