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Journal of Clinical Microbiology, July 2002, p. 2545-2554, Vol. 40, No. 7
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.7.2545-2554.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Molecular Peculiarities of the lytA Gene Isolated from Clinical Pneumococcal Strains That Are Bile Insoluble

Virginia Obregón,1 Pedro García,1 Ernesto García,1 Asunción Fenoll,2 Rubens López,1* and José L. García1

Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas,1 Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain2

Received 16 January 2002/ Returned for modification 10 April 2002/ Accepted 27 April 2002

The autolytic LytA amidase from 12 bile (deoxycholate)-insoluble streptococcal isolates (formerly classified as atypical Streptococcus pneumoniae) showing different antibiotic resistance patterns was studied. These atypical strains, which autolyze at the end of the stationary phase of growth, contain highly divergent lytA alleles (pairwise evolutionary distances of about 20%) compared to the lytA alleles of typical pneumococci. The atypical LytA amidases exhibit a peculiar deletion of two amino acids responsible for cell wall anchoring in the carboxy-terminal domain and have a reduced specific activity. These enzymes were inhibited by 1% deoxycholate but were activated by 1% Triton X-100, a detergent that could be used as an alternative diagnostic test for this kind of strain. Preparation of functional chimeric enzymes, PCR mutagenesis, and gene replacements demonstrated that the characteristic bile insolubility of these atypical strains was due to their peculiar carboxy-terminal domain and that the 2-amino-acid deletion was responsible for the inhibitory effect of deoxycholate. However, the deletion alone did not affect the specific activity of LytA. A detailed characterization of the genes encoding the 16S rRNA and SodA together with multilocus sequence typing indicated that the strains studied here are not a single clone and, although they cannot be strictly classified as typical pneumococci, they represent a quite diverse pool of organisms closely related to S. pneumoniae. The clinical importance of these findings is underlined by the role of the lytA gene in shaping the course of pneumococcal diseases. This study can also contribute to solving diagnostic problems and to understanding the evolution and pathogenic potential of species of the Streptococcus mitis group.

* Corresponding author. Mailing address: Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Velázquez 144, 28006 Madrid, Spain. Phone: (34) 91 561 1800. Fax: (34) 91 562 7518. E-mail: ruben{at}cib.csic.es.

Journal of Clinical Microbiology, July 2002, p. 2545-2554, Vol. 40, No. 7
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.7.2545-2554.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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