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Many bacterial species actively take up and recombine homologous DNA into their genomes, called natural competence, a trait that offers a means to identify the genetic basis of naturally occurring phenotypic variation. Here, we describe “transformed recombinant enrichment profiling” (TREP), in which natural transformation is used to generate complex pools of recombinants, phenotypic selection is used to enrich for specific recombinants, and deep sequencing is used to survey for the genetic variation responsible. We applied TREP to investigate the genetic architecture of intracellular invasion by the human pathogen  Haemophilus influenzae , a trait implicated in persistence during chronic infection. TREP identified the HMW1 adhesin as a crucial factor. Natural transformation of the  hmw1  operon from a clinical isolate (86-028NP) into a laboratory isolate that lacks it (Rd KW20) resulted in ~1,000-fold increased invasion into airway epithelial cells. When a distinct recipient (Hi375, already possessing  hmw1  and its paralog  hmw2 ) was transformed by the same donor, allelic replacement of  hmw2A    Hi375   by  hmw1A    86-028NP   resulted in a ~100-fold increased intracellular invasion rate. The specific role of  hmw1A    86-028NP   was confirmed by mutant and western blot analyses. Bacterial self-aggregation and adherence to airway cells were also increased in recombinants, suggesting that the high invasiveness induced by  hmw1A    86-028NP   might be a consequence of these phenotypes. However, immunofluorescence results found that intracellular  hmw1A    86-028NP   bacteria likely invaded as groups, instead of as individual bacterial cells, indicating an emergent invasion-specific consequence of  hmw1A -mediated self-aggregation.

Transformed Recombinant Enrichment Profiling Rapidly Identifies HMW1 as an Intracellular Invasion Locus in Haemophilus influenzae