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Journal of Bacteriology, June 2008, p. 4129-4138, Vol. 190, No. 12
0021-9193/08/$08.00+0     doi:10.1128/JB.01991-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Mutations in the tacF Gene of Clinical Strains and Laboratory Transformants of Streptococcus pneumoniae: Impact on Choline Auxotrophy and Growth Rate ^,

Ana González, Daniel Llull, María Morales, Pedro García, and Ernesto García^*

Centro de Investigaciones Biológicas (CSIC) and CIBER de Enfermedades Respiratorias (CIBERES), Ramiro de Maeztu 9, 28040 Madrid, Spain

Received 21 December 2007/ Accepted 8 April 2008

The nutritional requirement that Streptococcus pneumoniae has for the aminoalcohol choline as a component of teichoic and lipoteichoic acids appears to be exclusive to this prokaryote. A mutation in the tacF gene, which putatively encodes an integral membrane protein (possibly, a teichoic acid repeat unit transporter), has been recently identified as responsible for generating a choline-independent phenotype of S. pneumoniae (M. Damjanovic, A. S. Kharat, A. Eberhardt, A. Tomasz, and W. Vollmer, J. Bacteriol. 189:7105-7111, 2007). We now report that Streptococcus mitis can grow in choline-free medium, as previously illustrated for Streptococcus oralis. While we confirmed the finding by Damjanovic et al. of the involvement of TacF in the choline dependence of the pneumococcus, the genetic transformation of S. pneumoniae R6 by using S. mitis SK598 DNA and several PCR-amplified tacF fragments suggested that a minimum of two mutations were required to confer improved fitness to choline-independent S. pneumoniae mutants. This conclusion is supported by sequencing results also reported here that indicate that a spontaneous mutant of S. pneumoniae (strain JY2190) able to proliferate in the absence of choline (or analogs) is also a double mutant for the tacF gene. Microscopic observations and competition experiments during the cocultivation of choline-independent strains confirmed that a minimum of two amino acid changes were required to confer improved fitness to choline-independent pneumococcal strains when growing in medium lacking any aminoalcohol. Our results suggest complex relationships among the different regions of the TacF teichoic acid repeat unit transporter.

Published ahead of print on 18 April 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

These authors contributed equally to this work.