Hydroxyproline (Hyp) in decaying organic matter is a rich source of carbon and nitrogen for microorganisms. A bacterial pathway for Hyp catabolism is known; however, genes and function relationships are not established. In the pathway,
trans‐4‐hydroxy‐ l‐proline (4‐ l‐Hyp) is epimerized to cis‐4‐hydroxy‐ d‐proline (4‐ d‐Hyp), and then, in three enzymatic reactions, the d‐isomer is converted via Δ‐pyrroline‐4‐hydroxy‐2‐carboxylate (HPC) and α‐ketoglutarate semialdehyde (KGSA) to α‐ketoglutarate (KG). Here a transcriptional analysis of cells growing on 4‐ l‐Hyp, and the regulation and functions of genes from a Hyp catabolism locus of the legume endosymbiont Sinorhizobium melilotiare reported. Fourteen hydroxy proline catabolism genes ( hyp), in five transcripts hypR, hypD, hypH, hypSTand hypMNPQO(RE)XYZ, were negatively regulated by hypR. hypREwas shown to encode 4‐hydroxyproline 2‐epimerase and a hypREmutant grew with 4‐ d‐Hyp but not 4‐ l‐Hyp. hypO, hypDand hypHare predicted to encode 4‐ d‐Hyp oxidase, HPC deaminase and α‐KGSA dehydrogenase respectively. The functions for hypS, hypT, hypX, hypYand hypZremain to be determined. The data suggest 4‐Hyp is converted to the tricarboxylic acid cycle intermediate α‐ketoglutarate via the pathway established biochemically for Pseudomonas. This report describes the first molecular characterization of a Hyp catabolism locus.