Phenotype profiling of Rhizobium leguminosarum bv. trifolii clover nodule isolates reveal their both versatile and specialized metabolic capabilities

Andrzej Mazur 0 1 2 Graz_ yna Stasiak 0 1 2 Jerzy Wielbo 0 1 2 Piotr Koper 0 1 2 Agnieszka Kubik-Komar 0 1 2 Anna Skorupska 0 1 2 0 A. Kubik-Komar Chair of Applied Mathematics and Informatics, Lublin University of Life Sciences , Akademicka 13, 20-950 Lublin, Poland 1 A. Mazur (&) G. Stasiak J. Wielbo P. Koper A. Skorupska Department of Genetics and Microbiology, Maria Curie-Skodowska University , Akademicka 19, 20-033 Lublin, Poland 2 Communicated by Ursula Priefer Rhizobium leguminosarum bv. trifolii (Rlt) are soil bacteria inducing nodules on clover, where they fix nitrogen. Genome organization analyses of 22 Rlt clover nodule isolates showed that they contained 3-6 plasmids and majority of them possessed large ([1 Mb), chromidlike replicon with exception of four Rlt strains. The Biolog phenotypic profiling comprising utilization of C, N, P, and S sources and tolerance to osmolytes and pH revealed metabolic versatility of the Rlt strains. Statistical analyses of our results showed a clear bias toward specific metabolic preferences, tolerance to unfavorable osmotic conditions, and increased nodulation activity of the strains having smaller amount of extrachromosomal DNA. The K5.4 and K4.15 lacking a large megaplasmid possessed substantially diverse metabolism and belonged to effective clover inoculants. In conclusion, besides overall metabolic versatility, some metabolic specialization may enable rhizobia Andrzej Mazur and Graz_yna Stasiak are contributed equally to this work. - The rhizosphere is the microbe-rich zone surrounding plant roots. It is a dynamic environment, where resource distribution varies spatially and temporally, with plants providing a plethora of carbon and energy sources that significantly affect the populations of microorganisms in a manner specific to the host (Bais et al. 2006; Bertin et al. 2003; Haichar et al. 2008; Ramachandran et al. 2011). Bacteria must have evolved a wide variety of metabolic strategies to cope with such a dynamic environment. Moreover, they are often faced with unfavorable conditions such as osmotic stress, drought, heavy metals, and other toxins, as well as temperature changes. It is assumed that the structural and functional diversity of microbial communities in the rhizosphere is influenced by many biotic and abiotic factors (Berg and Smalla 2009). Rhizobia are an example of bacteria, which can survive in the soil where resources are scarce and diverse, and compete for nutrients with other bacteria present in the host plant rhizosphere (El Yahyaoui et al. 2004; Prell and Poole 2006). They can also enter into a beneficial symbiosis with legumes in a highly specialized environmentthe plant cell (Cai et al. 2009; Duodu et al. 2009; Faure et al. 2009). Rhizobia form nodules on the roots of their host legume plants. In exchange for carbohydrates provided by the plant, they fix atmospheric nitrogen and deliver reduced nitrogen compounds to their host (Gibson et al. 2008). Since rhizobia are found in different and complex environments, such as the soil, the rhizosphere, or plant cells, it is expected that they are capable of utilizing many different compounds. Soil bacteria (such as rhizobia) have complex and large, [6 Mb, genomes that reflect their diverse metabolic capabilities (Konstantinidis and Tiedje 2004). Such genomes are presumably ecologically advantageous in challenging environments. Thus, genome size and content could largely result from environmental pressure and bacterial adaptation to soil conditions (Barnett and Fisher 2006; Bentley and Parkhill 2004; Konstantinidis and Tiedje 2004; MacLean et al. 2007). The sequenced rhizobial genomes usually consist of a single circular chromosome and a set of plasmids, whose size ranges from several kb to Mb (Barran et al. 2001; Galibert et al. 2001; Gonzalez et al. 2006; Reeve et al. 2010a, b; Watson and Heys 2006; Young et al. 2006). Genomic content of rhizobia can be divided into two groups: the core genome, comprising genes present in all strains, and the accessory genome, consisting of unique or strain-specific genes (Young et al. 2006). The accessory genome comprises genes responsible for the symbiotic interaction with legume plants, which are typically located on one of the plasmids, called the symbiotic plasmid, or incorporated into the bacterial chromosome as symbiotic islands (Palacios and Newton 2005; Sullivan et al. 2002). Recently, in some rhizobia and other bacteria, extrachromosomal replicons called chromids were reported, with intermediate characteristics of the chromosome and plasmids (Harrison et al. 2010). Chromids are secondary replicons with plasmid maintenance and replication systems but bear some core genes and a far higher number of accessory genes than the chromosome. These genes are shared by chromids of other species in the same genus (Harrison et al. 2010). Rhizobium leguminosarum bv. trifolii (Rlt) is a microsymbiont of clover and is able to fix atmospheric nitrogen in root nodules of this plant. Our previous studies of Rlt isolates from root nodules of clover plants growing at the same site showed a substantial divergence of their genome organization, especially as regards the plasmid DNA content (Mazur et al. 2011). The isolates harbored between 3 and 6 plasmids with sizes from ca. 150 to 1,380 kb. The total approximated amount of extrachromosomal DNA in the sampled Rlt strains ranged from 1,890 (e.g., K3.6) kb to 3,250 kb (e.g., K4.13). Furthermore, most of the strains had large ([1 Mb), chromid-like replicon with the exception of four Rlt strains K3.6, K3.16, K4.15, and K5.4, in which this type of replicon was substantially smaller (Mazur et al. 2011). Despite the high variability in the number and size of plasmids in the studied strains, conservation of the location as well as the dynamic distribution of the individual genes (especially replication genes) in a specific genome compartment were demonstrated. Sequence divergence of particular genes was linked with their location in a given genome compartment, that is, the chromosome, chromid-like replicons, and plasmids. We also showed that the plasmid genes were less adapted to the host genome than the chromosome and the chromid-like genes (Mazur et al. 2011). Currently, the knowledge of how this genomic diversity is correlated with phenotype differentiation and strains adaptation to the challenging environment is fragmentary; however, a number of highthroughput phenotype arrays are being used for functional characterization of genes of model bacteria (AbuOun et al. 2009; Rodrigues et al. 2011; Sabarly et al. 2011). In the previous studies of metabolic variability within the Rlt strains, we have demonstrated a prevalence of metabolically versatile strains, that is, not specializing in utilization of any group of carbon sources (Wielbo et al. 2010). Metabolic versatility as regards nutritional requirements was not directly advantageous for effectiveness in the symbiotic intera (...truncated)