We report that catabolism of L-lactate in Bacillus subtilis depends on the previously uncharacterized yvfVyvfW-yvbY (herein renamed lutABC) operon, which is inferred to encode three iron-sulfur-containing proteins. The operon is under the dual control of a GntR-type repressor (LutR, formerly YvfI) and the master regulator for biofilm formation SinR and is induced during growth in response to L-lactate. Operons with high similarity to lutABC are present in the genomes of a variety of gram-positive and gram-negative bacteria, raising the possibility that LutABC is a widely conserved and previously unrecognized pathway for the utilization of L-lactate or related metabolites.The spore-forming bacterium Bacillus subtilis is capable of forming complex multicellular communities on surfaces (8,25,30,40). These communities, known as biofilms, consist of long chains of cells that are held together by an extracellular matrix consisting of protein and polysaccharide (9,31,36,37). Production of the matrix is governed by a complex regulatory network, at the heart of which are two parallel pathways of repression and antirepression (4,(10)(11)(12)27). One pathway involves the repressor AbrB, which controls the expression of many different kinds of genes, including genes involved in biofilm formation, during the transition from exponential growth to stationary phase (4,12,24). Relief from AbrBmediated repression is brought about in part by the antirepressor AbbA, which binds to and inactivates the repressor (4). The other pathway, consisting of the repressor SinR and its antirepressor SinI, is dedicated largely to genes involved in biofilm formation (10)(11)(12)27). The principal targets of the SinR repressor are the 18 genes of the epsA-to-O and yqxM-sipW-tasA operons, which are responsible for the production of the matrix (7,11,27,32). Several other genes and operons that are not directly required for matrix production are also under the control of the SinI-SinR pathway (11), one of which, the yvfVyvfW-yvbY operon, is the subject of this report.The initial goal of the current investigation was to elucidate the role, if any, of the yvfV-yvfW-yvbY operon, whose protein products were of unknown function, in biofilm formation. As we report herein, a clue as to the function of the operon came from comparative genomics, which led to the discovery that the yvfV-yvfW-yvbY operon specifies a pathway for the utilization of L-lactate. Here we demonstrate that the operon is required for growth on L-lactate as a sole carbon source; that it is subject to dual regulation, which allows it to be induced during both growth in liquid culture and biofilm formation; and that the operon influences the architectural complexity of biofilms formed in the presence of L-lactate. We therefore rename yvfV, yvfW, and yvbY as lutA, lutB, and lutC, respectively (for lactate utilization). Interestingly, homologous operons of lutABC are found in the genomes of many different bacteria, including some only distantly related to B. subtilis. These observations sug...