Sinorhizobium meliloti genome sequence determination has provided the basis for different approaches of functional genomics for this symbiotic nitrogen-fixing alpha-proteobacterium. One of these approaches is gene disruption with subsequent analysis of mutant phenotypes. This method is efficient for single genes; however, it is laborious and time-consuming if it is used on a large scale. Here, we used a signature-tagged transposon mutagenesis method that allowed analysis of the survival and competitiveness of many mutants in a single experiment. A novel set of signature tags characterized by similar melting temperatures and G؉C contents of the tag sequences was developed. The efficiencies of amplification of all tags were expected to be similar. Thus, no preselection of the tags was necessary to create a library of 412 signature-tagged transposons. To achieve high specificity of tag detection, each transposon was bar coded by two signature tags. In order to generate defined, nonredundant sets of signature-tagged S. meliloti mutants for subsequent experiments, 12,000 mutants were constructed, and insertion sites for more than 5,000 mutants were determined. One set consisting of 378 mutants was used in a validation experiment to identify mutants showing altered growth patterns.Sinorhizobium meliloti is a model organism for studies of plant-microbe interactions. This gram-negative soil bacterium can enter an endosymbiosis with alfalfa plants through the formation of nitrogen-fixing nodules. The availability of the 6.7-Mb S. meliloti genome sequence, which consists of one chromosome (3.65 Mb) and two megaplasmids, pSymA (1.36 Mb) and pSymB (1.68 Mb) (16), has enabled transcriptome (5, 32), proteome (14), and metabolome (6) studies. These approaches focus on the monitoring of RNA, protein, and metabolite levels. Moreover, a library of mobilizable plasmids carrying all open reading frames of this microorganism has been constructed (36). Another step toward a better functional understanding of the S. meliloti genome is the creation of large libraries of defined mutants by site-directed or random mutagenesis. Such mutant libraries can be used to study each mutant's phenotype under defined conditions. Usually, selection of mutants that can survive under certain conditions is simple and efficient and can be performed using a mixture of different mutants. However, selection of mutants that have an attenuated phenotype in test conditions is problematic, because all mutants have to be checked one by one. A microarray-based signature-tagged mutagenesis (STM) strategy (20; for reviews see references 4, 11, 19, 29, 34, and 38) can overcome this problem.Signature-tagged mutagenesis is based on a collection of mutants split into sets, in which each mutant is modified by one or more different signature tags. The tags are short DNA segments that are unique for each mutant in a set and can be amplified using invariant (for a review see reference 11) or specific (26) priming sites. Tagged mutants from the same set are pooled prior t...