title: Identification, distribution, and expression of novel genes in 10 clinical isolates of nontypeable Haemophilus influenzae.
abstract: We hypothesize that Haemophilus influenzae, as a species, possesses a much greater number of genes than that found in any single H. influenzae genome. This supragenome is distributed throughout naturally occurring infectious populations, and new strains arise through autocompetence and autotransformation systems. The effect is that H. influenzae populations can readily adapt to environmental stressors. The supragenome hypothesis predicts that significant differences exist between and among the genomes of individual infectious strains of nontypeable H. influenzae (NTHi). To test this prediction, we obtained 10 low-passage NTHi clinical isolates from the middle ear effusions of patients with chronic otitis media. DNA sequencing was performed with 771 clones chosen at random from a pooled genomic library. Homology searching demonstrated that approximately 10% of these clones were novel compared to the H. influenzae Rd KW20 genome, and most of them did not match any DNA sequence in GenBank. Amino acid homology searches using hypothetical translations of the open reading frames revealed homologies to a variety of proteins, including bacterial virulence factors not previously identified in the NTHi isolates. The distribution and expression of 53 of these genes among the 10 strains were determined by PCR- and reverse transcription PCR-based analyses. These unique genes were nonuniformly distributed among the 10 isolates, and transcription of these genes in planktonic cultures was detected in 50% (177 of 352) of the occurrences. All of the novel sequences were transcribed in one or more of the NTHi isolates. Seventeen percent (9 of 53) of the novel genes were identified in all 10 NTHi strains, with each of the remaining 44 being present in only a subset of the strains. These genic distribution analyses were more effective as a strain discrimination tool than either multilocus sequence typing or 23S ribosomal gene typing methods.
authors.affiliation: Center for Genomic Sciences, Allegheny-Singer Research Institute, Allegheny General Hospital, 320 East North Ave., 11th Floor South Tower, Pittsburgh, PA 15212, USA.
authors.forename: Kai
authors.initials: K
authors.lastname: Shen
authors.forename: Patricia
authors.initials: P
authors.lastname: Antalis
authors.forename: John
authors.initials: J
authors.lastname: Gladitz
authors.forename: Sameera
authors.initials: S
authors.lastname: Sayeed
authors.forename: Azad
authors.initials: A
authors.lastname: Ahmed
authors.forename: Shujun
authors.initials: S
authors.lastname: Yu
authors.forename: Jay
authors.initials: J
authors.lastname: Hayes
authors.forename: Sandra
authors.initials: S
authors.lastname: Johnson
authors.forename: Bethany
authors.initials: B
authors.lastname: Dice
authors.forename: Richard
authors.initials: R
authors.lastname: Dopico
authors.forename: Randy
authors.initials: R
authors.lastname: Keefe
authors.forename: Benjamin
authors.initials: B
authors.lastname: Janto
authors.forename: William
authors.initials: W
authors.lastname: Chong
authors.forename: Joseph
authors.initials: J
authors.lastname: Goodwin
authors.forename: Robert M
authors.initials: RM
authors.lastname: Wadowsky
authors.forename: Geza
authors.initials: G
authors.lastname: Erdos
authors.forename: J Christopher
authors.initials: JC
authors.lastname: Post
authors.forename: Garth D
authors.initials: GD
authors.lastname: Ehrlich
authors.forename: Fen Z
authors.initials: FZ
authors.lastname: Hu
mesh_terms: D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry; D016680:Genome, Bacterial; D044404:Genomic Islands; D006193:Haemophilus influenzae / Q000145:classification / Q000235:genetics* / Q000472:pathogenicity; D006801:Humans; D008969:Molecular Sequence Data; D010802:Phylogeny; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D020449:Repetitive Sequences, Amino Acid; D014774:Virulence
chemical_list: D004269:DNA, Bacterial; D012338:RNA, Ribosomal, 23S