Nontypeable Haemophilus influenzae (NTHi) is a significant human pathogen as it is a major cause of otitis media (OM), sinusitis, exacerbations of chronic obstructive pulmonary disease (COPD) and bronchitis. Illness due to NTHi is thus a major societal burden, and an aggressive approach is needed to determine novel ways of combating diseases caused by NTHi. In all aspects of NTHi disease, the organism moves from being a passive member of the upper respiratory tract flora to an opportunistic pathogen in the site of infection. Although iron and heme uptake systems have been identified in NTHi, little is known about how iron and heme uptake is regulated. Also, NTHi is protected from the damage caused by excess iron or heme that is extremely toxic, primarily through production of toxic reactive oxygen species via the Fenton reaction. The mechanisms involved in this protection are unknown. We have used cDNA microarrays to interrogate the transcriptome of strain 86-028NP, an NTHi strain whose genome we have sequenced. We also have constructed mutants in strain 86-028NP that are deficient in the expression of the oxyR gene as well as the fur (ferric uptake regulator) gene and identified the genes in the OxyR and Fur regulons. We propose to further characterize the response of NTHi strain 86-028NP to oxidative stress. Emphasis will be placed on the interrelationship between oxidative stress and control of iron and heme uptake and usage. The fur regulon will be further characterized. Experiments are proposed to identify and characterize Fur- and oxidative stress- regulated small RNAs. Mutants deficient in the expression of both Fur- and sRNA-regulated genes of unknown or poorly defined function as well as genes encoding proteins involved in resistance to oxidative stress will be constructed and characterized. The NTHi strain 86-028NP heme oxygenase will be identified and characterized. These experiments will lead to an improved understanding of how NTHi regulates its iron and heme metabolism and will provide insight into previously uncharacterized iron-regulated genes, some of which may be involved in heme storage and/or heme degradation. The proposed studies will also provide data on the interplay between the oxidative stress response systems and the Fur system in NTHi thereby increasing our understanding of how NTHi responds to environmental signals and providing us with information that will be useful in the design of new approaches to prevent disease caused by NTHi.