Gram-negative bacteria display either a flat or an irregular outer membrane. The periodontal pathogen Aggregatibacter (Actinobacillus) actinomycetemcomitans has an irregular outer membrane. We have identified a gene that is associated with the biogenesis of this morphology. The gene is part of a three-gene operon and codes for a 141-kDa protein designated morphogenesis protein C (MorC), which is conserved in several gram-negative bacteria including Haemophilus influenzae and Pasteurella multocida. Insertional inactivation of this gene resulted in the conversion of an irregularly shaped membrane to a flat membrane. Associated with this morphological change were the autoaggregation of the bacteria during planktonic growth and a concomitant increase in the surface hydrophobicity of the bacterium. The absence of MorC also resulted in the loss of the secretion of leukotoxin but not the ltxA transcription. Our findings suggest that MorC is critical for membrane morphology and leukotoxin secretion in A. actinomycetemcomitans.Aggregatibacter (Actinobacillus) actinomycetemcomitans is a small gram-negative, nonmotile, coccobacillus important in the pathogenesis of localized aggressive periodontitis and some cases of adult periodontitis (43,53). A. actinomycetemcomitans also translocates from the oral cavity into the circulatory system to cause extraoral infections, including infective endocarditis and abscesses in various body sites (34,35). A. actinomycetemcomitans is a member of the clinically relevant HACEK group of pathogens, consisting of Haemophilus species (H. parainfluenzae, H. aphrophilus, and H. paraphrophilus), A. actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella species, which has an enhanced capacity to produce endocardial infections (5). Recently, we demonstrated the interaction of A. actinomycetemcomitans with cardiac valves and the role of this organism in a rabbit endocarditis animal model (49).Isolates of A. actinomycetemcomitans from the oral cavity typically display long bundled fimbriae (17,38,42). In broth cultures, these rough strains autoaggregate and adhere to the walls of the growth vessel. During planktonic growth, small cohorts of bacteria lose the ability to express fimbriae and adopt a smooth phenotype (18,20). These cells (smooth strains) grow homogenously in broth cultures and demonstrate minimal adherence to glass surfaces. Transmission electron microscopy (TEM) studies reveal distinctive membrane morphologies for both rough and smooth variants (6, 28). The bacterial outer membrane of both phenotypes has an irregular or rugose appearance. This membrane morphology is associated with some gram-negative bacteria, e.g., H. influenzae (2) and Moraxella catarrhalis (16), but not with others, e.g., Escherichia coli, Salmonella spp., and Yersinia spp., which have flat outer membranes. The physiological relevance and biogenesis of these membrane convolutions have not been investigated. The outer membrane as a whole is critical for the physiology and protection of gra...