Cyclic di-AMP targets the cystathionine beta-synthase domain of the osmolyte transporter OpuC

Abstract

Cellular turgor is of fundamental importance to bacterial growth and survival. Changes in external osmolarity as a consequence of fluctuating environmental conditions and colonization of diverse environments can significantly impact cytoplasmic water content, resulting in cellular lysis or plasmolysis. To ensure maintenance of appropriate cellular turgor, bacteria import ions and small organic osmolytes, deemed compatible solutes, to equilibrate cytoplasmic osmolarity with the extracellular environment. Here, we show that elevated levels of c-di-AMP, a ubiquitous second messenger among bacteria, result in significant susceptibility to elevated osmotic stress in the bacterial pathogen Listeria monocytogenes. We found that levels of import of the compatible solute carnitine show an inverse correlation with intracellular c-di-AMP content and that c-di-AMP directly binds to the CBS domain of the ATPase subunit of the carnitine importer OpuC. Biochemical and structural studies identify conserved residues required for this interaction and transport activity in bacterial cells. Overall, these studies reveal a role for c-di-AMP mediated regulation of compatible solute import and provide new insight into the molecular mechanisms by which this essential second messenger impacts bacterial physiology and adaptation to changing environmental conditions.

Figure 1

Inverse correlation between c-di-AMP levels and osmotic stress tolerance. (A) Phenotype microarray results of WT vs. ΔpdeA ΔpgpH on PM9 wells A1–A9 with increasing NaCl concentrations. (B) The L. monocytogenes cΔdacA, wild-type (WT), and ΔpdeA ΔpgpH strains were tested for growth in BHI broth with increasing NaCl concentrations. One-hundred percent indicates growth rate for each strain in the absence of NaCl. (C) Similar to (B) but growth rates were examined in increasing sorbitol concentrations. (D) Growth yields in minimal medium with no salt, or minimal medium + 2% NaCl (MMS) with or without betaine, carnitine, and potassium chloride at indicated concentrations.

Figure 2

Carnitine transport under salt stress in the presence of different c-di-AMP levels. (A) Mid-exponential phase cultures grown in BHI broth were shifted to BHI + 4% NaCl + 85 μM ¹⁴C- carnitine. Imported carnitine was evaluated by radioactivity retained by bacterial cells, and normalized to optical density at each time point. (B) Plot comparing the intracellular c-di-AMP levels of various L. monocytogenes strains versus the amount of carnitine transported at 20 minutes in panel A. The WT c-di-AMP level is typically 4 μM. Data represents at least two independent experiments.

Figure 3

C-di-AMP interacts specifically with the CBS domain of the carnitine transporter component OpuCA. (A) Domain organization of OpuCA (Lmo1428), GbuA (Lmo1014), and BilE (Lmo1421). (B) Binding of OpuCA, GbuA, and BilE to c-di-AMP as measured by DRaCALA to full-length (FL), ATP-binding (ATP), or CBS domains. (C) Binding titration for the OpuCA full-length OpuCA protein and (D) the isolated CBS domain. (E) Competition binding assay for ~1.5 nM radiolabeled c-di-AMP, in the presence of 200 μM unlabeled nucleotides. (C) Binding titration for the E. faecalis full-length OpuCA homolog.

Figure 4

Crystal structure of LmOpuCA tandem CBS domains in complex with c-di-AMP. (A) Schematic drawing of the dimer of LmOpuCA tandem CBS domains (monomer 1 in cyan, monomer 2 in yellow) in complex with c-di-AMP (labeled cdA, carbon atoms colored black). The two CBS domains in each monomer are labeled CBS1 and CBS2. (B) Omit F_o–F_c electron density for c-di-AMP at 2.9 Å resolution, contoured at 2.5σ. (C) Molecular surface of the LmOpuCA binding sites for c-di-AMP, colored according to the electrostatic potential (blue: positive, red: negative). (D) Detailed interactions between LmOpuCA and c-di-AMP. Hydrogen bonds are shown with dashed lines (red). (E) Sequence conservation of residues in the LmOpuCA c-di-AMP binding site, generated based on an alignment of 150 sequences by the program Consurf (Armon et al., 2001). Purple indicates conserved residues, cyan indicates variable residues, and white indicates average conservation. All structure figures were produced with PyMOL (www.pymol.org).

Figure 5

C-di-AMP binding mutants exhibit altered carnitine transport activities. (A) Binding curves of OpuCA CBS point mutants with radiolabeled c-di-AMP (B) Effects of opuCA mutant alleles in the ΔpdeA ΔpgpH background on carnitine transport. (C) Plot comparing the amount of ¹⁴C-carnitine transported in panel B at 30 minutes versus the observed binding constant for c-di-AMP. V280A and D281A K_d values were above the limit of detection of the binding assay. Data represents at least two independent experiments.