Background: The regulation of cell wall hydrolysis by the pneumococcal autolysin LytA is poorly understood.Results: The cell wall is susceptible to extracellular LytA only during the stationary phase or after cell wall synthesis inhibition.Conclusion: LytA is regulated on the substrate level, where peptidoglycan modifications likely prevent LytA hydrolysis.Significance: The control of amidases is essential for bacterial survival, cell-wall synthesis, and division.

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SummaryThe molecular mechanisms underlying cell growth, cell division and pathogenesis in Streptococcus pneumoniae are still not fully understood. Single-cell methodologies are potentially of great value to investigate S. pneumoniae cell biology. Here, we report the construction of novel plasmids for single and double cross-over integration of functional fusions to the gene encoding a fast folding variant of the green fluorescent protein (GFP) into the S. pneumoniae chromosome. We have also established a zincinducible system for the fine control of gfp-fusion gene expression and for protein depletion experiments in S. pneumoniae. Using this novel single cell toolkit, we have examined the cellular localization of the proteins involved in the essential process of choline decoration of S. pneumoniae teichoic acid. GFP fusions to LicA and LicC, enzymes involved in the activation of choline, showed a cytoplasmic distribution, as predicted from their primary sequences. A GFP fusion to the choline importer protein LicB showed clear membrane localization. GFP fusions to LicD1 and LicD2, enzymes responsible for loading of teichoic acid subunits with choline, are also membrane-associated, even though both proteins lack any obvious membrane spanning domain. These results indicate that the decoration of teichoic acid by the LicD enzymes is a membrane-associated process presumably occurring at lipid-linked teichoic acid precursors.

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 G3T 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.One of the unique properties of the human pathogen Streptococcus pneumoniae is the strict nutritional requirement for choline (18), which is taken up from the growth medium and metabolized exclusively to be attached to the wall teichoic acid and the lipoteichoic acid (25). The choline residues of the teichoic acid are involved in a wide variety of physiological functions, including the activation of the murein (peptidoglycan) hydrolases LytA, LytB, and LytC (8,9,11,17), the binding of a class of surface proteins (the choline-binding proteins) (20,29), and the interaction with bacteriophages (15) and host proteins, for example, with the receptor for the platelet-activating factor (5). The essentiality of choline residues for pneumococcal virulence recently has been demonstrated (13).Choline is transported into the cytoplasm and is activated to CDP-choline by the products of the licABC genes (2, 3). CDPcholine is the substrate of proteins (presumably the products of the licD1 and licD2 genes) that attach the phosphoryl choline residues to the teichoic acid precursors (30). These genes are clustered in the lic region on the pneumococcal chromosome, which contains three additional genes, spr1148, spr1149, and spr1150, of unknown functions. Based on sequence comparisons, it was suggested that these genes may encode an alcohol dehydrogenase (spr1148), a CDP-ribitol pyrophosphorylase (spr1149), and a sugar transporter (spr1150) (30).Although the cell wall choline residues are of great importance for the physiology of S. pneumoniae, none of the cholinerelated functions is essential for cell growth, and the molecular mechanism(s) responsible for the choline dependency of pneumococcal g...