ABSTRACT Rhizobium sp. strain NGR234 is a unique alphaproteobacterium (order Rhizobiales ) that forms nitrogen-fixing nodules with more legumes than any other microsymbiont. Since we have previously described the complete genome sequence of NGR234, we now report on a genome-wide functional analysis of the genes and enzymes involved in autoinducer I hydrolysis in this microbe. Altogether we identified five cosmid clones that repeatedly gave a positive result in our function-based approach for the detection of autoinducer I hydrolase genes. Of these five cosmid clones, two were located on pNGR234 b and three were on cNGR234. Subcloning and in vitro mutagenesis in combination with BLAST analyses identified the corresponding open reading frames (ORFs) of all cosmid clones: dlhR , qsdR1 , qsdR2 , aldR , and hitR-hydR . Analyses of recombinant DlhR and QsdR1 proteins by using high-performance liquid chromatography-mass spectrometry (HPLC-MS) demonstrate that these enzymes function as acyl homoserine lactone (AHL) lactonases. Furthermore, we showed that these enzymes inhibited biofilm formation and other quorum-sensing-dependent processes in Pseudomonas aeruginosa , Chromobacterium violaceum , and Agrobacterium tumefaciens . Finally, our experimental data suggest that competitive colonization of roots in the rhizospheres of cowpea plants is affected by DlhR and QsdR1.