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Objective To investigate reciprocal regulation between Fur and two RyhB homologs in Yersinia pestis (Y. pestis), as well as the roles of RyhBs in biofilm formation. Methods Regulatory relationships were assessed by a combination of colony morphology assay, primer extension, electrophoretic mobility shift assay and DNase I footprinting. Results Fur bound to the promoter-proximal DNA regions of ryhB1 and ryhB2 to repress their transcription, while both RyhB1 and RyhB2 repressed the expression of Fur at the post-transcriptional level. In addition, both RyhB1 and RyhB2 positively regulated Y. pestis biofilm exopolysaccharide (EPS) production and the expression of hmsHFRS and hmsT. Conclusion Fur and the two RyhB homologs exert negative reciprocal regulation, and RyhB homologs have a positive regulatory effect on biofilm formation in Y. pestis.