ABSTRACTDimethyl sulfide (DMS) is a volatile sulfur compound produced mainly from the degradation of dimethylsulfoniopropionate (DMSP) in marine environments. DMS undergoes oxidation to form dimethyl sulfoxide (DMSO), dimethyl sulfone (DMSO2), and methanesulfonate (MSA), all of which occur in terrestrial environments and are accessible for consumption by various microorganisms. The purpose of the present study was to determine how the enhancer-binding proteins SfnR1 and SfnR2 contribute to the utilization of DMS and its derivatives inPseudomonas aeruginosaPAO1. First, results from cell growth experiments showed that deletion of eithersfnR2orsfnG, a gene encoding a DMSO2-monooxygenase, significantly inhibits the ability ofP. aeruginosaPAO1 to use DMSP, DMS, DMSO, and DMSO2as sulfur sources. Deletion of thesfnR1ormsuEDCgenes, which encode a MSA desulfurization pathway, did not abolish the growth ofP. aeruginosaPAO1 on any sulfur compound tested. Second, data collected from β-galactosidase assays revealed that themsuEDC-sfnR1operon and thesfnGgene are induced in response to sulfur limitation or nonpreferred sulfur sources, such as DMSP, DMS, and DMSO, etc. Importantly, SfnR2 (and not SfnR1) is essential for this induction. Expression ofsfnR2is induced under sulfur limitation but independently of SfnR1 or SfnR2. Finally, the results of this study suggest that the main function of SfnR2 is to direct the initial activation of themsuEDC-sfnR1operon in response to sulfur limitation or nonpreferred sulfur sources. Once expressed, SfnR1 contributes to the expression ofmsuEDC-sfnR1,sfnG, and other target genes involved in DMS-related metabolism inP. aeruginosaPAO1.IMPORTANCEDimethyl sulfide (DMS) is an important environmental source of sulfur, carbon, and/or energy for microorganisms. For various bacteria, includingPseudomonas,Xanthomonas, andAzotobacter, DMS utilization is thought to be controlled by the transcriptional regulator SfnR. Adding more complexity, some bacteria, such asAcinetobacter baumannii,Enterobacter cloacae, andPseudomonas aeruginosa, possess two, nonidentical SfnR proteins. In this study, we demonstrate that SfnR2 and not SfnR1 is the principal regulator of DMS metabolism inP. aeruginosaPAO1. Results suggest that SfnR1 has a supportive but nonessential role in the positive regulation of genes required for DMS utilization. This study not only enhances our understanding of SfnR regulation but, importantly, also provides a framework for addressing gene regulation through dual SfnR proteins in other bacteria.