Construction of a Large Signature-Tagged Mini-Tn 5 Transposon Library and Its Application to Mutagenesis of Sinorhizobium meliloti

Abstract

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...

The RpiR-Like Repressor IolR Regulates Inositol Catabolism in Sinorhizobium meliloti

“…The iolR, iolD, and iolE mutants of S. meliloti 2011 contain mTn5-STM::gusA insertions, obtained by transposon mini-Tn5 signature-tagged mutagenesis (mTn5-STM), in the same orientation as the respective genes, creating transcriptional fusions that allow the investigation of iolRDE gene expression by determining the ␤-glucuronidase activity (27). (The mTn5-STM mutation in the iolC mutant is oriented in the opposite direction as the iolC gene, and no transposon-induced iolB mutant is available in S. meliloti strain 2011.…”

Identification of Mesorhizobium loti Genes Relevant to Symbiosis by Using Signature-Tagged Mutants

“…One of the differences in symbiosis between M. loti and S. meliloti is that the latter bacteroid undergoes terminal differentiation through endoreduplication, causing it to lose reproductive capacity, whereas the former maintains reproductive capacity during symbiosis (28). Therefore, it is significant that large-scale mutant libraries of mapped transposon insertion mutants have been constructed to enable STM screening of both M. loti and S. meliloti (34,41). Complementary functional genomics in these two species should provide a complete picture of the symbiosis between rhizobia and legumes.…”

“…STM has since been applied to a number of pathogenic or symbiotic bacteria (for a review, see reference 27). An STM library was constructed for the symbiotic bacterium S. meliloti by using 412 transposons with unique tags that can be detected by hybridization to microarrays (34). A pilot screening of this library showed that a number of genes relevant to symbiosis or competition could be identified (35).…”

RNase E and RNase J are needed for S-adenosylmethionine homeostasis in Sinorhizobium meliloti

“…We used the S. meliloti strain Rm2011 (referred to here as strain 2011; [48,49] and its RNase E and RNase J mutants 2011rne :: Tn5 and 2011rnj :: Tn5, which harbour mini-Tn5 insertions in the respective genes rne (SMc01336) and rnj (SMc01929) [7,31,37]. The mutant strains are referred to here as 2011rne and 2011rnj, respectively.…”

“…The role of S. meliloti RNase J in rRNA maturation was studied using a mutant with a mini-Tn5 insertion destroying the only one rnj gene [31,37]. The rRNA maturation defect of this RNase J mutant was complemented perfectly by constitutive ectopic rnj expression [31].…”