Molecular and structural basis of glutathione import in Gram-positive bacteria via GshT and the cystine ABC importer TcyBC of Streptococcus mutans

Glutathione (GSH) protects cells against oxidative injury and maintains a range of vital functions across all branches of life. Despite recent advances in our understanding of the transport mechanisms responsible for maintaining the spatiotemporal homeostasis of GSH and its conjugates in eukaryotes and Gram-negative bacteria, the molecular and structural basis of GSH import into Gram-positive bacteria has remained largely uncharacterized. Here, we employ genetic, biochemical and structural studies to investigate a possible glutathione import axis in Streptococcus mutans, an organism that has hitherto served as a model system. We show that GshT, a type 3 solute binding protein, displays physiologically relevant affinity for GSH and glutathione disulfide (GSSG). The crystal structure of GshT in complex with GSSG reveals a collapsed structure whereby the GS-Ileg of GSSG is accommodated tightly via extensive interactions contributed by the N-and C-terminal lobes of GshT, while the GS-II leg extends to the solvent. This can explain the ligand promiscuity of GshT in terms of binding glutathione analogues with substitutions at the cysteine-sulfur or the glycine-carboxylate. Finally, we show that GshT primes glutathione import via the L-cystine ABC transporter TcyBC, a membrane permease, which had previously exclusively been associated with the transport of L-cystine

Characterization of a Glutamate Transporter Operon, glnQHMP, in Streptococcus mutans and Its Role in Acid Tolerance▿ †

Glutamate contributes to the acid tolerance response (ATR) of many Gram-negative and Gram-positive bacteria, but its role in the ATR of the oral bacterium Streptococcus mutans is unknown. This study describes the discovery and characterization of a glutamate transporter operon designated glnQHMP (Smu.1519 to Smu.1522) and investigates its potential role in acid tolerance. Deletion of glnQHMP resulted in a 95% reduction in transport of radiolabeled glutamate compared to the wild-type UA159 strain. The addition of glutamate to metabolizing UA159 cells resulted in an increased production of acidic end products, whereas the glnQHMP mutant produced less lactic acid than UA159, suggesting a link between glutamate metabolism and acid production and possible acid tolerance. To investigate this possibility, we conducted a microarray analysis with glutamate and under pH 5.5 and pH 7.5 conditions which showed that expression of the glnQHMP operon was downregulated by both glutamate and mild acid. We also measured the growth kinetics of UA159 and its glnQHMP-negative derivative at pH 5.5 and found that the mutant doubled at a much slower rate than the parent strain but survived at pH 3.5 significantly better than the wild type. Taken together, these findings support the involvement of the glutamate transporter operon glnQHMP in the acid tolerance response in S. mutans

The overall structure of Sgo0707N.

<p>A: Ribbon representation of Sgo0707N. The N1-domain is coloured pink and the N2-domain purple. B: Topology diagram of Sgo0707N1. C: Topology diagram of Sgo0707N2. β-strands are depicted as triangles and α-helices as circles. The N1- and N2-domains are shown in pink and purple respectively.</p

Role of Sgo0707 in <i>S. gordonii</i> binding to type-1 collagen, saliva, serum and oral keratinocytes.

<p>A: Fluorescence micrographs showing adherence of wild-type and ΔSgo0707 strains to protein-coated surfaces and to oral keratinocytes. In panels 1–3 bacteria were flowed over surfaces coated with type-1 collagen, 25% saliva or 10% serum for 2 h, as described, and adhered bacteria stained green using the LIVE/DEAD® <i>Bac</i>light™ Bacterial Viability stain. Images were obtained using CLSM and the surface coverage determined by image analysis using the <i>bio</i>Image_L software package. In panel 4, bacteria were flowed over keratinocyte layers for 1 h, stained using the LIVE <i>Bac</i>Light™ Bacterial Gram Stain Kit and viewed with CLSM. The mean number of bacteria (stained red)/1000 keratinocytes (stained green) was determined by manual image analysis. B: Graphs showing the adherence of the wild-type (grey bars) and ΔSgo0707 (white bars) strains to different surface coatings. For type-1 collagen, saliva and serum, adherence is expressed as surface coverage (arbitrary units) whereas for the keratinocytes, binding is expressed as number of bacteria/1000 keratinocytes. The mean and standard error of three independent experiments is shown (* indicates a significant difference p<0.05).</p

Data collection, refinement and model quality statistics for Sgo0707.

a<p>Values in parentheses indicate statistics for the highest resolution shell.</p>b<p><i>R</i>_sym = Σ<i>_hkl</i> Σ<i>_i</i> |<i>I</i>_i<i>(hkl)</i> - <<i>I</i>(<i>hkl</i>)>|/Σ<i>_hkl</i> Σ<i>_i I</i>_i (<i>hkl</i>), where <i>I_i</i>(<i>hkl</i>) is the intensity of the <i>i</i>th observation of reflection <i>hkl and </i> is the average over of all observations of reflection <i>hkl.</i>^c<i>R</i>_work = Σ | |<i>F</i>_obs| - | <i>F</i>_calc| |/Σ | <i>F</i>_obs|, where <i>F</i>_obs and <i>F</i>_calc are the observed and calculated structure factor amplitudes, respectively. <i>R</i>_free is <i>R</i>_work calculated using 5% of the data, randomly omitted from refinement.</p

Sgo0707 collagen docking.

<p>A model of collagen docked to the Sgo0707 crystal structure using Firedock. A: The collagen triple helix is docked into the binding pocket of the N1-domain. B: Collagen is docked in the groove formed by the interface between the N1- and N2-domains.</p

Protein melting temperatures when supplemented with metal ion solutions.

<p>Protein melting temperatures when supplemented with metal ion solutions.</p

Summary of the VicR consensus sequences as determined by DNaseI footprinting.

<p>Summary of the VicR consensus sequences as determined by DNaseI footprinting.</p

The putative binding cleft in the N1-domain.

<p>A: Stereo representation of the cleft in the N1-domain. Amino acids in the pocket are represented as cylinders and are labeled. B: The N1-domain is presented as an electrostatic surface, colored in red and blue according to negative and positive electrostatic potential, respectively. The cleft has considerable negative electrostatic potential. The figure is shown in stereo.</p