We report the identification of a previously unknown Listeria monocytogenes gene, flaR, which modulates DNA topology. Through the analysis of a Tn917 nonmotile mutant, LOSC1, in which production of flagellin was abolished, we have identified a bacterial component involved in gene regulation. The transposon had inserted in flaR, an open reading frame of 531 bp, followed by a second open reading frame of 1252 bp in reverse orientation. On the L. monocytogenes physical map, flaR was located in a different region from that of the flaA gene encoding flagellin. Transcriptional analysis showed that the flaR gene product affects the flaA expression and negatively regulates its own expression. When expressed in Escherichia coli, flaR encodes a protein of 18 kDa (FlaR) whose transcription is osmoregulated. In addition, FlaR also influences the expression of reporter genes containing supercoiling-sensitive promoters such as proU or ompC. The data presented here suggest that FlaR is a histone-like bacterial protein which acts at specific sites to influence DNA topology and, therefore, transcription. flaR is the first gene of this class to be described in Gram-positive pathogenic bacteria.