Caulobacter crescentus, which thrives in freshwater environments with low nutrient levels, serves as a model system for studying bacterial cell cycle regulation and organelle development. We examined its ability to utilize lactose (i) to gain insight into the metabolic capacities of oligotrophic bacteria and (ii) to obtain an additional genetic tool for studying this model organism, aiming to eliminate the basal enzymatic activity that hydrolyzes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X-gal). Using a previously isolated transposon mutant, we identified a gene, lacA, that is required for growth on lactose as the sole carbon source and for turning colonies blue in the presence of X-gal. LacA, which contains a glucose-methanol-choline (GMC) oxidoreductase domain, has homology to the flavin subunit of Pectobacterium cypripedii's gluconate dehydrogenase. Sequence comparisons indicated that two genes near lacA, lacB and lacC, encode the other subunits of the membrane-bound dehydrogenase. In addition to lactose, all three lac genes are involved in the catabolism of three other ␤-galactosides (lactulose, lactitol, and methyl-␤-D-galactoside) and two glucosides (salicin and trehalose). Dehydrogenase assays confirmed that the lac gene products oxidize lactose, salicin, and trehalose. This enzymatic activity is inducible, and increased lac expression in the presence of lactose and salicin likely contributes to the induction. Expression of lacA also depends on the presence of the lac genes, implying that the dehydrogenase participates in induction. The involvement of a dehydrogenase suggests that degradation of lactose and other sugars in C. crescentus may resemble a proposed pathway in Agrobacterium tumefaciens.Caulobacter species inhabit diverse aquatic and soil environments, their ubiquitous distribution reflecting an ability to prosper despite low nutrient levels (47, 48). Unraveling the physiological adaptations that enable these aerobic chemoheterotrophs to thrive under nutrient-poor conditions will facilitate interpretation of microbial activities in oligotrophic environments, such as water bodies (which make up the majority of the earth's surface). Recent studies of Caulobacter have led to novel findings regarding the uptake and catabolism of various carbon sources (6,14,38,44,52). In addition, one particular member of the group, Caulobacter crescentus, has emerged as a prominent model for the study of bacterial cell cycle progression, asymmetric cell division, and organelle development (7,8,34,49). Although factors that regulate the C. crescentus cell cycle appear to respond to changes in metabolic states (3,22,35), details of the connection remain nebulous. Investigation of C. crescentus's metabolic capabilities can improve understanding of bacterial cell cycle control as well as the biogeochemical contributions of oligotrophs.We initiated an examination of C. crescentus's ability to utilize lactose for two reasons. (i) C. crescentus cells predominantly dwell in freshwater pond...