The essential tacF gene is responsible for the choline-dependent growth phenotype of Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae has an absolute nutritional requirement for choline, and the choline molecules are known to incorporate exclusively into the cell wall and membrane teichoic acids of the bacterium. We describe here the isolation of a mutant of strain R6 in which a single G-->T point mutation in the gene tacF (formerly designated spr1150) is responsible for generating a choline-independent phenotype. The choline-independent phenotype could be transferred to the laboratory strain R6 and to the encapsulated strain D39 by genetic transformation with a PCR product or with a plasmid carrying the mutated tacF gene. The tacF gene product belongs to the protein family of polysaccharide transmembrane transporters (flippases). A model is presented in which TacF is required for the transport of the teichoic acid subunits across the cytoplasmic membrane. According to this model, wild-type TacF has a strict specificity for choline-containing subunits, whereas the TacF present in the choline-independent mutant strain is able to transport both choline-containing and choline-free teichoic acid chains. The proposed transport specificity of parental-type TacF for choline-containing subunits would ensure the loading of the cell wall with teichoic acid chains decorated with choline residues, which appear to be essential for the virulence of this pathogen.

FIG. 1.

Organization of the lic region of the different strains used in this study. The base at position 700 of the spr1150 (tacF) gene is indicated. An asterisk indicates an inactivated licA gene. Divergent transcription of the choline utilization genes driven by promoters is denoted by a perpendicular arrow before spr1149 and spr1150, respectively. erm, erythromycin resistance gene present on the integration plasmid pJDC9. Phenotype abbreviations: D, choline-dependent growth; I, choline-independent growth; +, choline incorporation from the growth medium; −, no choline incorporation from the growth medium.

FIG. 2.

Growth of the different strains on Cden plates with or without choline and erythromycin. Only the strains with the mutated tacF gene, R6Chi and R6pJDC1150M, are capable of growing in the absence of choline. Only the strains transformed with a pJDC9-based plasmid are resistant to erythromycin.

FIG. 3.

Cells of R6Chi and R6pJDC1150M visualized by light microscopy. Both strains grew as diplococci, and they grew in short chains in the presence of choline and in long chains in the absence of choline. Bar, 10 μm.

FIG. 4.

Virulence of choline-free S. pneumoniae in the mouse intraperitoneal model. Bacteria were injected into the intraperitoneal cavity at different cell concentrations, and the survival of mice was monitored for 3 to 7 days. Strain D39Chip was inoculated with 10³ CFU/mouse (solid lines with solid squares), and strain D39ChiplicB31 (dashed lines and open symbols) was inoculated with the following concentrations (in CFU/mouse): 10³ (open triangles), 10⁵ (open circles), 10⁶ (open diamonds), and 10⁷ (open squares).

FIG. 5.

Model for the specificity of the proposed TacF teichoic acid subunit flippase and for the choline-dependent growth phenotype. (A) The wild-type TacF(V²³⁴) flippase has a strict specificity for choline-containing teichoic acid subunits and does not transport subunits without choline, ensuring the loading of the cell wall with choline residues (left side). The TacF(F²³⁴) flippase, encoded by the mutated tacF gene in R6Chi, is able to transport both choline-containing and choline-free teichoic acid subunits. RU, repeating unit; PCho, phosphoryl choline; black bar, lipid anchor. (B) The membrane steps of teichoic acid and peptidoglycan biosynthesis pathways are interconnected by the undecaprenyl phosphate transport lipid (black rectangle), which is released in the diphosphate form during polymerization of the peptidoglycan (PG) and teichoic acid (TA) strands, respectively, and then is recycled for further cycles of subunit transport. (C) In the absence of choline, wild-type cells cannot produce teichoic acids because of the specificity of TacF for choline-loaded teichoic acid subunits. (D) The mutant strain R6Chi is able to grow in the absence of choline and to produce a choline-free cell wall, because the altered flippase (TacF*) has lost its specificity for choline-containing subunits.