Generation and Properties of a Streptococcus pneumoniae Mutant Which Does Not Require Choline or Analogs for Growth

A mutant (JY2190) of Streptococcus pneumoniae Rx1 which had acquired the ability to grow in the absence of choline and analogs was isolated. Lipoteichoic acid (LTA) and wall teichoic acid (TA) isolated from the mutant were free of phosphocholine and other phosphorylated amino alcohols. Both polymers showed an unaltered chain structure and, in the case of LTA, an unchanged glycolipid anchor. The cell wall composition was also not altered except that, due to the lack of phosphocholine, the phosphate content of cell walls was half that of the parent strain. Isolated cell walls of the mutant were resistant to hydrolysis by pneumococcal autolysin (N-acetylmuramyl-l-alanine amidase) but were cleaved by the muramidases CPL and cellosyl. The lack of active autolysin in the mutant cells became apparent by impaired cell separation at the end of cell division and by resistance against stationary-phase and penicillin-induced lysis. As a result of the absence of choline in the LTA, pneumococcal surface protein A (PspA) was no longer retained on the cytoplasmic membrane. During growth in the presence of choline, which was incorporated as phosphocholine into LTA and TA, the mutant cells separated normally, did not release PspA, and became penicillin sensitive. However, even under these conditions, they did not lyse in the stationary phase, and they showed poor reactivity with antibody to phosphocholine and an increased release of C-polysaccharide from the cell. In contrast to ethanolamine-grown parent cells (A. Tomasz, Proc. Natl. Acad. Sci. USA 59:86–93, 1968), the choline-free mutant cells retained the capability to undergo genetic transformation but, compared to Rx1, with lower frequency and at an earlier stage of growth. The properties of the mutant could be transferred to the parent strain by DNA of the mutant.Pneumococci differ from other gram-positive bacteria in that their lipoteichoic acid (LTA) and wall teichoic acid (TA) have the same chain structure which is, moreover, unusually complex (Fig. ​(Fig.1):1): glycerophosphate is replaced by ribitol phosphate (7), and between the ribitol phosphate residues a tetrasaccharide is intercalated (23). It contains d-glucose, 2-acetamido-4-amino-2,4,6-trideoxy-d-galactose (AATGal), and two N-acetyl-d-galactosaminyl residues, one or both of which carry a phosphocholine residue at O-6 (references 3 and 12 and this report). Open in a separate windowFIG. 1Pneumococcal TA and LTA. As shown, in strain R6 most of the repeats carry two phosphocholine residues each, at O-6 of the N-acetyl-d-galactosaminyl residues (3, 12). In strain Rx1 and Rx1/AL⁻, most repeats contain one phosphocholine residue (this report) attached to O-6 of the non-ribitol-linked galactosaminyl residue (14).Pneumococci are not able to synthesize the choline required for the synthesis of these substituents. Moreover, choline is an essential growth factor (2, 30) but can be substituted in this function by nutritional ethanolamine (EA) (38). Phosphoethanolamine is incorporated into LTA and TA in place of phosphocholine (14), but it cannot replace phosphocholine functionally. Phosphocholine-substituted LTA serves to anchor pneumococcal surface protein A (PspA) to the outer layer of the cytoplasmic membrane, with choline-mediated interaction between membrane-associated LTA and the C-terminal repeat region of PspA. In EA-grown bacteria, PspA is no longer retained and is released into the surrounding medium (45). Phosphocholine substituents also play an essential role for the activity of the major pneumococcal autolysin, an N-acetylmuramyl-l-alanine amidase (38). This protein possesses a choline-binding C-terminal domain that is essential for activity but, unlike PspA, is not essential for retention on the pneumococcal cell surface (16, 32). Binding of phosphocholine-substituted LTA to this domain results in potent inhibition of the amidase (21). The inhibitory property is dependent on the micellar structure of LTA (13) and lost by deacylation (5). Phosphocholine-substituted LTA may also participate in the transport of the amidase through the cytoplasmic membrane from the cytosol (5), the location of its synthesis (15). It additionally effects the conversion of the inactive E form of the enzyme into the active C form (5). This conversion is likewise effected by the choline residues of cell wall-linked TA (33, 39). Furthermore, binding of the amidase to the choline residues of TA is prerequisite for the hydrolysis of cell walls by the enzyme (18, 22). It should be noted that the amidase is not essential for growth. Though the enzyme is completely inactive in EA grown cells, the growth rate is not affected. However, cell separation is impaired, and there is a loss of stationary-phase and penicillin-induced cell lysis (38, 40), as well as a loss of genetic transformation (38). After insertional inactivation of the autolysin gene (lytA), the autolysin-deficient mutants (Lyt⁻) grew normally (31) and did not even show impeded cell separation (41).In this report, we describe a mutant which acquired the ability to grow in the absence of choline and analogs. Except for the observation that [³H]choline-substituted LTA is not a precursor of [³H]choline-substituted TA (6), nothing is known about the biosyntheses of pneumococcal LTA and TA and the stage of biosynthesis at which phosphocholine is incorporated. Since the absence of choline incorporation might affect the structure of LTA and TA as well as the composition of cell walls, we included relevant analyses in our study.(A preliminary report of this work was presented in an overview on pneumococcal LTA and TA at the International Meeting on the Molecular Biology of Streptococcus pneumoniae and Its Diseases, Oeiras, Portugal, September 24 to 29, 1996 [10].)