Abstract Haemophilus influenzae is a member of the normal bacterial flora colonizing the human nasopharynx. It is also a cause of infections of the upper respiratory tract, such as otitis media and bronchitis, and of systemic infections, such as bacteremia and meningitis. Ideally, prevention or treatment of H. influenzae disease would affect only disease-associated bacteria without perturbing normal flora. The long-term goal of this work is to understand the transition from asymptomatic colonization to mucosal or systemic infection. We hypothesize that one of the mechanisms by which nasopharyngeal bacteria adapt to a new environment such as the middle ear or the bloodstream is phase variation of a DNA methylase gene, mod. Phase variation of surface structures such as outer membrane proteins and lipopolysaccharide has been studied in H. influenzae for several years. The mod gene contains a tandem repeat region similar to those identified in other, well-characterized phase-variable genes in H. influenzae, but a physiologic role for mod phase variation was unknown until recently. Our collaborators compared a mod deletion mutant with a mod+ parent by microarray analysis and reported that mod affects transcription of at least 15 genes (Srikhanta et al. 2005 PNAS 102:5547-51) apparently via methylation of promoters. Not all of these genes have known functions, but generally genes upregulated by mod expression are involved in nutrient acquisition, while several of those downregulated by mod expression are in the heat-shock group. It is proposed that phase variation of mod is a mechanism for random, coordinated on/off switching of several other genes. These studies used the laboratory-adapted, avirulent strain Rd KW20. Phase-variable mod genes have also been identified in clinical isolates of H. influenzae and in other mucosal organisms such as Neisseria meningitidis. This exploratory project will study this novel mechanism of gene regulation in virulent H. influenzae, (i) using microarrays to identify mod-related genes in clinical isolates and (ii) examining the effect of mod phase in experimental infections. Finding that mod regulation is common in clinical isolates and affects pathogenesis will provide the basis for future studies on the physiological adaptation of bacteria to different anatomic niches within the human host.