Haemophilus influenzae has an absolute requirement for NAD (factor V) because it lacks all biosynthetic enzymes necessary for de novo synthesis of that cofactor. Therefore, growth in vitro requires the presence of NAD itself, NMN, or nicotinamide riboside (NR). To address uptake abilities of these compounds, we investigated outer membrane proteins. By analyzing ompP2 knockout mutants, we found that NAD and NMN uptake was prevented, whereas NR uptake was not. Through investigation of the properties of purified OmpP2 in artificial lipid membrane systems, the substrate specificity of OmpP2 for NAD and NMN was determined, with K S values of ϳ8 and 4 mM, respectively, in 0.1 M KCl, whereas no interaction was detected for the nucleoside NR and other purine or pyrimidine nucleotide or nucleoside species. Based on our analysis, we assume that an intrinsic binding site within OmpP2 exists that facilitates diffusion of these compounds across the outer membrane, recognizing carbonyl and exposed phosphate groups. Because OmpP2 was formerly described as a general diffusion porin, an additional property of acting as a facilitator for nicotinamide-based nucleotide transport may have evolved to support and optimize utilization of the essential cofactor sources NAD and NMN in H. influenzae.The only known natural habitat of Haemophilus influenzae is the human nasopharynx. H. influenzae is Gram-negative and is able to grow under facultative anaerobic conditions. It is also a human pathogen and is responsible for significant morbidity and mortality in young children (1, 2). To cultivate H. influenzae, a complex medium is required; and if not blood-based, it must contain two growth factors: NAD and hemin (3). In the nasopharynx, H. influenzae finds a relatively constant environment, and the organism has retained a certain degree of flexibility in its ability to utilize nutrients (4) by possessing some limiting metabolic pathway redundancy (5).Early biochemical investigations have established that NMN 1 and nicotinamide riboside (NR) can substitute for NAD, whereas nicotinamide, niacin, or other nicotine-based intermediates of the Preiss-Handler pathway cannot (6 -8). The NAD dependence of H. influenzae was confirmed by the absence of genes encoding the enzymes necessary for the de novo biosynthesis of NAD (9). Accumulation of nicotinamide nucleotides derived from NAD or NR has been demonstrated in H. influenzae and Haemophilus parainfluenzae (10, 11). For H. parainfluenzae, the K m for transport is ϳ0.55 M for NAD and 0.14 M for NR, and the V max for NR is about four times that of NAD (11). This implies that NR is the substrate for an as yet unidentified inner membrane transporter. Recently, we presented data showing that two gene products appear to be involved in the NAD utilization pathway of H. influenzae (12)(13)(14). The gene products were identified as the hel-encoded outer membrane lipoprotein e(P4) and a periplasmic protein termed NadN. Knockout mutations of both genes resulted in growth-deficient phenotypes depending on the...