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The distributed genome hypothesis (DGH) states that each strain within a bacterial species receives a unique distribution of genes from a population-based supragenome that is many times larger than the genome of any given strain. The observations that natural infecting populations are often polyclonal and that most chronic bacterial pathogens have highly developed mechanisms for horizontal gene transfer suggested the DGH and provided the means and the mechanisms to explain how chronic infections persist in the face of a mammalian host's adaptive defense mechanisms. Having previously established the validity of the DGH for obligate pathogens, we wished to evaluate its applicability to an opportunistic bacterial pathogen. This was accomplished by construction and analysis of a highly redundant pooled genomic library containing approximately 216,000 functional clones that was constructed from 12 low-passage clinical isolates of Pseudomonas aeruginosa, 6 otorrheic isolates and 6 from other body sites. Sequence analysis of 3,214 randomly picked clones (mean insert size, approximately 1.4 kb) from this library demonstrated that 348 (10.8%) of the clones were unique with respect to all genomic sequences of the P. aeruginosa prototype strain, PAO1. Hypothetical translations of the open reading frames within these unique sequences demonstrated protein homologies to a number of bacterial virulence factors and other proteins not previously identified in P. aeruginosa. PCR and reverse transcription-PCR-based assays were performed to analyze the distribution and expression patterns of a 70-open reading frame subset of these sequences among 11 of the clinical strains. These sequences were unevenly distributed among the clinical isolates, with nearly half (34/70) of the novel sequences being present in only one or two of the individual strains. Expression profiling revealed that a vast majority of these sequences are expressed, strongly suggesting they encode functional proteins.

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 approximately 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.

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.