Kassapa Ellepola ¹ Lauren C Guillot ¹ Bradley J. Comeaux ¹ Yiran (Taylor) Han ² Jessica Kajfasz ³ Jacob P Bitoun ⁴ Grace Ann Spatafora ² Jose Lemos ⁵ Zezhang Tom Wen ^1*

• ¹ LSU Health Sciences Center New Orleans, Louisiana State University, New Orleans, United States
• ² Department of Biology, Middlebury College, Middlebury, VT, Vermont, United States
• ³ College of Dentistry, University of Florida, Gainesville, Florida, United States
• ⁴ Department of Microbiology and Immunology, School of Medicine, Tulane University, New Orleans, Louisiana, United States
• ⁵ Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, Florida, United States

The sufCDSUB gene cluster, encoding the sole iron-sulfur (Fe-S) cluster assembly system in S. 29 mutans, was recently shown to be up-regulated in response to oxidative stressors and Fe 30 limitation. In this study, luciferase reporter fusion assays, electrophoretic gel mobility shift 31 assays (EMSA) and in vitro transcription assays (IVT) were used to dissect the cis-and trans-32 acting factors that regulate the expression of sufCDSUB. Results showed deletion of perR, for the 33 only Fur-family transcriptional regulator in S. mutans, resulted in >5-fold increases in luciferase 34 activity under the control of the sufCDSUB promoter (P<0.01), as compared to the parent strain, 35 UA159 when the reporter strains were grown in medium with no supplemental iron. Site-directed 36 mutagenesis of a PerR-box in the promoter region led to elevation of the reporter activity by 37 >1.6-fold (P<0.01). In an EMSA, recombinant PerR (rPerR) was shown to bind to the cognate 38 sufCDSUB promoter leading to mobility retardation. On the other hand, the reporter activity was 39 increased by >84-fold (P<0.001) in response to the addition of cysteine at 4 mM to the culture 40 medium. Deletion of cysR, for a LysR-type of transcriptional regulator, led to reduction of the 41 reporter activity by >11.6-fold (P<0.001). Addition of recombinant CysR (rCysR) to an EMSA 42 caused mobility shift of the sufCDSUB promoter probe, indicative of rCysR-promoter 43 interaction, and rCysR was shown to enhance sufC transcription under the direction of 44 sufCDSUB promoter in vitro. These results suggest that multiple factors are involved in the 45 regulation of sufCDSUB expression in response to environmental cues, including cysteine and Fe 46 availability, consistent with the important role of sufCDSUB in S. mutans physiology.