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Infection and Immunity, January 2006, p. 321-330, Vol. 74, No. 1
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.1.321-330.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Characterization, Distribution, and Expression of Novel Genes among Eight Clinical Isolates of Streptococcus pneumoniae {dagger}

Kai Shen,1 John Gladitz,1 Patricia Antalis,1 Bethany Dice,1 Benjamin Janto,1 Randy Keefe,1 Jay Hayes,1 Azad Ahmed,1 Richard Dopico,1 Nathan Ehrlich,1,{ddagger} Jennifer Jocz,1,§ Laura Kropp,1,¶ Shujun Yu,1 Laura Nistico,1 David P. Greenberg,3 Karen Barbadora,3 Robert A. Preston,1 J. Christopher Post,1,2 Garth D. Ehrlich,1,2* and Fen Z. Hu1,2

Center for Genomic Sciences, Allegheny-Singer Research Institute, Pittsburgh, Pennsylvania 15212,1 Department of Microbiology and Immunology, Drexel University College of Medicine, Allegheny Campus, Pittsburgh, Pennsylvania 15212,2 Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania 152133

Received 1 May 2005/ Returned for modification 13 July 2005/ Accepted 21 September 2005

Eight low-passage-number Streptococcus pneumoniae clinical isolates, each of a different serotype and a different multilocus sequence type, were obtained from pediatric participants in a pneumococcal vaccine trial. Comparative genomic analyses were performed with these strains and two S. pneumoniae reference strains. Individual genomic libraries were constructed for each of the eight clinical isolates, with an average insert size of ~1 kb. A total of 73,728 clones were picked for arraying, providing more than four times genomic coverage per strain. A subset of 4,793 clones were sequenced, for which homology searches revealed that 750 (15.6%) of the sequences were unique with respect to the TIGR4 reference genome and 263 (5.5%) clones were unrelated to any available streptococcal sequence. Hypothetical translations of the open reading frames identified within these novel sequences showed homologies to a variety of proteins, including bacterial virulence factors not previously identified in S. pneumoniae. The distribution and expression patterns of 58 of these novel sequences among the eight clinical isolates were analyzed by PCR- and reverse transcriptase PCR-based analyses, respectively. These unique sequences were nonuniformly distributed among the eight isolates, and transcription of these genes in planktonic cultures was detected in 81% (172/212) of their genic occurrences. All 58 novel sequences were transcribed in one or more of the clinical strains, suggesting that they all correspond to functional genes. Sixty-five percent (38/58) of these sequences were found in 50% or less of the clinical strains, indicating a significant degree of genomic plasticity among natural isolates.

* Corresponding author. Mailing address: Center for Genomic Sciences, Allegheny-Singer Research Institute, Allegheny General Hospital, 320 East North Ave., 11th Floor South Tower, Pittsburgh, PA 15212. Phone: (412) 359-4228. Fax: (412) 359-6995. E-mail: gehrlich{at}wpahs.org.

Editor: J. N. Weiser

{dagger} Supplemental material for this article may be found at http://iai.asm.org/.

{ddagger} Present address: University of Pittsburgh, Pittsburgh, PA 15213.

§ Present address: Carnegie-Mellon University, Pittsburgh, PA 15213.

Present address: Pennsylvania State University, State College, PA 16802.

Infection and Immunity, January 2006, p. 321-330, Vol. 74, No. 1
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.1.321-330.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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