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Biochemical and spectroscopic properties of Brucella microti glutamate decarboxylase, a key component of the glutamate-dependent acid resistance system.

Grassini G, Pennacchietti E, Cappadocio F, Occhialini A, De Biase D - FEBS Open Bio (2015)

Bottom Line: BmGadB is hexameric at acidic pH.On the contrary, cellular localization analysis, corroborated by sequence inspection, suggests that BmGadB does not undergo membrane recruitment at acidic pH, which was observed in EcGadB.The comparison of GadB from evolutionary distant microorganisms suggests that for this enzyme to be functional in GDAR some structural features must be preserved.

View Article: PubMed Central - PubMed

Affiliation: Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, 04100 Latina, Italy.

ABSTRACT
In orally acquired bacteria, the ability to counteract extreme acid stress (pH ⩽ 2.5) ensures survival during transit through the animal host stomach. In several neutralophilic bacteria, the glutamate-dependent acid resistance system (GDAR) is the most efficient molecular system in conferring protection from acid stress. In Escherichia coli its structural components are either of the two glutamate decarboxylase isoforms (GadA, GadB) and the antiporter, GadC, which imports glutamate and exports γ-aminobutyrate, the decarboxylation product. The system works by consuming protons intracellularly, as part of the decarboxylation reaction, and exporting positive charges via the antiporter. Herein, biochemical and spectroscopic properties of GadB from Brucella microti (BmGadB), a Brucella species which possesses GDAR, are described. B. microti belongs to a group of lately described and atypical brucellae that possess functional gadB and gadC genes, unlike the most well-known "classical" Brucella species, which include important human pathogens. BmGadB is hexameric at acidic pH. The pH-dependent spectroscopic properties and activity profile, combined with in silico sequence comparison with E. coli GadB (EcGadB), suggest that BmGadB has the necessary structural requirements for the binding of activating chloride ions at acidic pH and for the closure of its active site at neutral pH. On the contrary, cellular localization analysis, corroborated by sequence inspection, suggests that BmGadB does not undergo membrane recruitment at acidic pH, which was observed in EcGadB. The comparison of GadB from evolutionary distant microorganisms suggests that for this enzyme to be functional in GDAR some structural features must be preserved.

No MeSH data available.


Related in: MedlinePlus

pH dependent cellular partition in EcGadB (left panel) and BmGadB (right panel). 12% SDS–PAGE of 30 μg-samples of cell supernatants (S), obtained after cell lysis, cytoplasmic (C) and membrane (M) fractions obtained as described in Section 2.4 at neutral (pH 7.2) and mildly acidic pH (pH 5.5 EcGadB or pH 5.1 BmGadB). The region of the gel encompassing the bands corresponding to EcGadB and BmGadB is shown with a blue box. Molecular weight (kDa) standards are shown on the right of the left panel. The decarboxylase activity is provided as percentage with respect to the corresponding starting activity in S (100%). The reported activity values represent the mean of 2–3 independent experiments, with a standard deviation not exceeding 10% of the stated value.
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f0035: pH dependent cellular partition in EcGadB (left panel) and BmGadB (right panel). 12% SDS–PAGE of 30 μg-samples of cell supernatants (S), obtained after cell lysis, cytoplasmic (C) and membrane (M) fractions obtained as described in Section 2.4 at neutral (pH 7.2) and mildly acidic pH (pH 5.5 EcGadB or pH 5.1 BmGadB). The region of the gel encompassing the bands corresponding to EcGadB and BmGadB is shown with a blue box. Molecular weight (kDa) standards are shown on the right of the left panel. The decarboxylase activity is provided as percentage with respect to the corresponding starting activity in S (100%). The reported activity values represent the mean of 2–3 independent experiments, with a standard deviation not exceeding 10% of the stated value.

Mentions: Secondary structure prediction tools indicate that the N-terminal sequence of BmGadB (which contains a Pro residue at position 7) does not support formation of α-helices. Conversely, the same tools predict the N-terminal region of EcGadB to be able to form α-helices and this agrees with the crystal structure data [18]. Despite the apparent inability to form an α-helix in the N-terminal region, BmGadB might still retain the ability to partition between the cytosol and the membrane following a decrease in cytoplasmic pH. In order to address this point, cell fractionation of the E. coli BL21(DE3) overexpressing BmGadB was carried out. Cytoplasmic and membrane fractions at neutral pH and at pH 5.1, in the presence of chloride, were assayed for enzyme activity and analyzed by SDS–PAGE (Fig. 7). The same analysis was carried out on an E. coli strain overexpressing EcGadB, as previously reported [18]. The pH 5.1 was chosen for BmGadB because at this pH the enzyme is still in the active form (Fig. 5 and Table 2). As predicted on the basis of the N-terminal amino acid sequence, the cellular localization of BmGadB is not influenced by a pH decrease, unlike that of EcGadB (Fig. 7 and [18]), thus suggesting that in BmGadB the N-terminal region does not undergo the same conformational changes which were reported to occur in EcGadB. Notably, a significant fraction of the protein (approximately 40%) is associated to the membrane already at neutral pH and no further recruitment to the membrane is observed when the pH of the cell extract is lowered to 5.1.


Biochemical and spectroscopic properties of Brucella microti glutamate decarboxylase, a key component of the glutamate-dependent acid resistance system.

Grassini G, Pennacchietti E, Cappadocio F, Occhialini A, De Biase D - FEBS Open Bio (2015)

pH dependent cellular partition in EcGadB (left panel) and BmGadB (right panel). 12% SDS–PAGE of 30 μg-samples of cell supernatants (S), obtained after cell lysis, cytoplasmic (C) and membrane (M) fractions obtained as described in Section 2.4 at neutral (pH 7.2) and mildly acidic pH (pH 5.5 EcGadB or pH 5.1 BmGadB). The region of the gel encompassing the bands corresponding to EcGadB and BmGadB is shown with a blue box. Molecular weight (kDa) standards are shown on the right of the left panel. The decarboxylase activity is provided as percentage with respect to the corresponding starting activity in S (100%). The reported activity values represent the mean of 2–3 independent experiments, with a standard deviation not exceeding 10% of the stated value.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4382515&req=5

f0035: pH dependent cellular partition in EcGadB (left panel) and BmGadB (right panel). 12% SDS–PAGE of 30 μg-samples of cell supernatants (S), obtained after cell lysis, cytoplasmic (C) and membrane (M) fractions obtained as described in Section 2.4 at neutral (pH 7.2) and mildly acidic pH (pH 5.5 EcGadB or pH 5.1 BmGadB). The region of the gel encompassing the bands corresponding to EcGadB and BmGadB is shown with a blue box. Molecular weight (kDa) standards are shown on the right of the left panel. The decarboxylase activity is provided as percentage with respect to the corresponding starting activity in S (100%). The reported activity values represent the mean of 2–3 independent experiments, with a standard deviation not exceeding 10% of the stated value.
Mentions: Secondary structure prediction tools indicate that the N-terminal sequence of BmGadB (which contains a Pro residue at position 7) does not support formation of α-helices. Conversely, the same tools predict the N-terminal region of EcGadB to be able to form α-helices and this agrees with the crystal structure data [18]. Despite the apparent inability to form an α-helix in the N-terminal region, BmGadB might still retain the ability to partition between the cytosol and the membrane following a decrease in cytoplasmic pH. In order to address this point, cell fractionation of the E. coli BL21(DE3) overexpressing BmGadB was carried out. Cytoplasmic and membrane fractions at neutral pH and at pH 5.1, in the presence of chloride, were assayed for enzyme activity and analyzed by SDS–PAGE (Fig. 7). The same analysis was carried out on an E. coli strain overexpressing EcGadB, as previously reported [18]. The pH 5.1 was chosen for BmGadB because at this pH the enzyme is still in the active form (Fig. 5 and Table 2). As predicted on the basis of the N-terminal amino acid sequence, the cellular localization of BmGadB is not influenced by a pH decrease, unlike that of EcGadB (Fig. 7 and [18]), thus suggesting that in BmGadB the N-terminal region does not undergo the same conformational changes which were reported to occur in EcGadB. Notably, a significant fraction of the protein (approximately 40%) is associated to the membrane already at neutral pH and no further recruitment to the membrane is observed when the pH of the cell extract is lowered to 5.1.

Bottom Line: BmGadB is hexameric at acidic pH.On the contrary, cellular localization analysis, corroborated by sequence inspection, suggests that BmGadB does not undergo membrane recruitment at acidic pH, which was observed in EcGadB.The comparison of GadB from evolutionary distant microorganisms suggests that for this enzyme to be functional in GDAR some structural features must be preserved.

View Article: PubMed Central - PubMed

Affiliation: Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, 04100 Latina, Italy.

ABSTRACT
In orally acquired bacteria, the ability to counteract extreme acid stress (pH ⩽ 2.5) ensures survival during transit through the animal host stomach. In several neutralophilic bacteria, the glutamate-dependent acid resistance system (GDAR) is the most efficient molecular system in conferring protection from acid stress. In Escherichia coli its structural components are either of the two glutamate decarboxylase isoforms (GadA, GadB) and the antiporter, GadC, which imports glutamate and exports γ-aminobutyrate, the decarboxylation product. The system works by consuming protons intracellularly, as part of the decarboxylation reaction, and exporting positive charges via the antiporter. Herein, biochemical and spectroscopic properties of GadB from Brucella microti (BmGadB), a Brucella species which possesses GDAR, are described. B. microti belongs to a group of lately described and atypical brucellae that possess functional gadB and gadC genes, unlike the most well-known "classical" Brucella species, which include important human pathogens. BmGadB is hexameric at acidic pH. The pH-dependent spectroscopic properties and activity profile, combined with in silico sequence comparison with E. coli GadB (EcGadB), suggest that BmGadB has the necessary structural requirements for the binding of activating chloride ions at acidic pH and for the closure of its active site at neutral pH. On the contrary, cellular localization analysis, corroborated by sequence inspection, suggests that BmGadB does not undergo membrane recruitment at acidic pH, which was observed in EcGadB. The comparison of GadB from evolutionary distant microorganisms suggests that for this enzyme to be functional in GDAR some structural features must be preserved.

No MeSH data available.


Related in: MedlinePlus