The ISME Journal (2008) 2, 158–170 & 2008 International Society for Microbial Ecology All rights reserved 1751-7362/08 $30.00 www.nature.com/ismej ORIGINAL ARTICLE Insights learned from pBTAi1, a 229-kb accessory plasmid from Bradyrhizobium sp. strain BTAi1 and prevalence of accessory plasmids in other Bradyrhizobium sp. strains Eddie J Cytryn1,2, Siriluck Jitacksorn1,2, Eric Giraud3 and Michael J Sadowsky1,2 1Department of Soil, Water, and Climate, BioTechnology Institute, University of Minnesota, St Paul, MN, USA; 2Microbial and Plant Genomics Institute, University of Minnesota, St Paul, MN, USA and 3Laboratoire des Symbioses Tropicales et Me´diterrane´ennes, IRD, Montpellier, France In silico, physiological and in planta analyses were used to characterize pBTAi1, a 229-kb accessory plasmid from Bradyrhizobium sp. strain BTAi1, and assess its potential ecological function under free-living and symbiotic growth conditions. Sequence analysis revealed the presence of an uptake hydrogenase system, a repABC family plasmid replication module and open reading frames encoding type IV secretion system, TraI and TraR autoinducer proteins and several copper À1 resistance-related proteins. Bradyrhizobium sp. BTAi1 was capable of growing in 200 mg l CuCl2. In contrast, the closely related, plasmid-free Bradyrhizobium sp. strain ORS278 could not grow at copper concentrations exceeding 100 mg lÀ1. The plasmid-localized hydrogenase genes were phylogenetically distinct from those typically found in other rhizobial species, and were most related to hup genes from Thiobacillus denitrificans. The induction of the plasmid-borne hydrogenase genes during symbiosis was significantly lower than the two chromosomal-borne hydrogenase clusters. CHEF-pulsed-field gel electrophoresis was used for a comprehensive analysis of the diversity, abundance and genetic composition of accessory plasmids in other Bradyrhizobium strains. Plasmids were detected in 11 of 46 (23.9%) geographically diverse Bradyrhizobium japonicum and Bradyrhizobium elkanii strains, isolated from the United States, China and Thailand. Plasmid size was heterogeneous, ranging from 75 to 330 kb, with only two strains (DASA01244 and DASA01265) harboring plasmids with identical (240 kb) size. None of the plasmids harbored nodulation or hydrogenase genes. Taken together, our results indicate that while plasmids having ecologically significant functions may be detected in Bradyrhizobium sp. strains, they lack genes necessary for symbioses with legumes. The ISME Journal (2008) 2, 158–170; doi:10.1038/ismej.2007.105; published online 24 January 2008 Subject Category: integrated genomics and post-genomics approaches in microbial ecology Keywords: genomics; plasmids; function; symbiosis; hydrogenase Introduction of growth on laboratory media: the fast growers, which consist of strains in the genera Rhizobium, The primary source of biologically-fixed nitrogen Sinorhizobium and Azorhizobium; the intermediate (N2) in agricultural systems is produced via the growth rate strains in the genus Mesorhizobium and symbiotic interaction between legumes and the the slow growers, which comprise strains in the root- and shoot-nodulating soil bacteria, collectively phylogenetically distinct genus Bradyrhizobium referred to as the rhizobia. The rhizobia can be (Sawada et al., 2003). divided into three broad groups based on their speed Fast- and slow-growing rhizobia are also unique with respect to the location of genes involved in nodulation and symbiotic nitrogen fixation. Correspondence: MJ Sadowsky, Department of Soil, Water, and Generally speaking, the structural genes for the Climate, BioTechnology Institute, University of Minnesota, 1991 nitrogenase enzyme (nifHDK) and genes essential Upper Buford Circle, 439 Borlaug Hall, St Paul, MN 55108, USA. E-mail: [email protected] for the production of lipochitooligosaccharide no- Received 27 August 2007; revised 13 October 2007; accepted 19 dulation factors (nodABC) in the fast-growing October 2007; published online 24 January 2008 species are located on large, high-molecular weight, Sequence and analysis of pBTAi1 EJ Cytryn et al 159 plasmids (pSym; Merrick and Edwards, 1995). DNA replicons in their host bacterium (Ramirez- Plasmids can vary significantly between and within Romero et al., 2000). species of rhizobia, ranging from 180 kb in Rhizo- In contrast to the significant number of reports of bium leguminosarum bv. trifolii, to 1.35 Mb in plasmids in the fast-growing rhizobia, there have Sinorhizobium meliloti (Mercado-Blanco and Toro, been relatively few studies concerning the presence 1996; Barnett et al., 2001). In contrast, genes of plasmids in Bradyrhizobium strains, perhaps due encoding nod factors and nitrogenase in Bradyrhi- to methodological limitations. While a few studies zobium species and most mesorhizobia are located have reported the presence of two to four plasmids on the chromosome in regions termed ‘symbiosis in B. japonicum strains, ranging in size from 74 to islands’ (Sawada et al., 2003). In the soybean 294 kb (Gross et al., 1979; Masterson et al., 1982), symbiont Bradyrhizobium japonicum, symbiosis- the taxonomic status of some of the strains was related and hydrogenase genes are localized to not verified using molecular, phylogenetic tools on a 611-kb chromosomal-borne symbiosis island (Gross et al., 1979; Masterson et al., 1982). Although that displays a distinct distribution of insertion Masterson et al. (1982) showed that nod genes were sequence elements relative to the rest of the not found on any plasmids they detected, no chromosome (Gottfert et al., 2001; Kaneko et al., information was presented regarding the genetic 2002). Interestingly, while Mesorhizobium loti has a composition of these plasmids, and thus the chromosomal-borne symbiosis island, the moder- physiological role of plasmids in bradyrhizobia ately slow-growing Mesorhizobium amorphae was could not be inferred. In addition, due to changes found to carry a 930-kb pSym (Wang et al., 1999). in the taxonomy of soybean-nodulating bacteria, Several Rhizobium and Bradyrhizobium species some strains have since been reclassified as belong- contain uptake hydrogenases, which reduce energy ing to Sinorhizobium fredii, a fast-growing species. loss associated with symbiotic nitrogen fixation To date, the functional role of plasmids in the (Albrecht et al., 1979; Evans et al., 1987). Generally, bradyrhizobia has not been explored, and until now the hydrogen uptake (hup) genes are arranged in at (Giraud et al., 2007) there have been no reports on least three operons with conserved gene composi- the complete sequence and analysis of plasmids tion and organization (Baginsky et al., 2002). The from any Bradyrhizobium strain. hupS and hupL genes encode for hydrogenase The Bradyrhizobium strain BTAi1 belongs to a structural subunits, whereas the remaining hup novel group of phototrophic bradyrhizobia that form and hyp gene products encode for maturation a symbiotic association with several species in the proteins, which are responsible for the recruitment legume genus Aeschynomene. Members of this and incorporation of nickel and other metals into bacterial group although taxonomically related to the hydrogenase active site (Casalot and Rousset, other bradyrhizobia, are unique in their ability to 2001). While the R. leguminosarum and B. japoni- couple photosynthesis to nitrogen fixation (Giraud cum hydrogenase systems are highly homologous, and Fleischman, 2004). Recently, it was reported they differ in gene regulation and in the presence of that Bradyrhizobium sp. strain BTAi1, and other specific genes. In Rhizobium tropici and R. legumi- bradyrhizobia capable of forming nodules on nosarum the hup genes are encoded on the Aeschynomene indica, are taxonomically related to symbiotic plasmid, whereas in B. japonicum, Blastobacter denitrificans, and it has been proposed Bradyrhizobium sp. (Lupinus) and Bradyrhizobium that the later bacterium be transferred to the genus sp. (Vigna), the hup genes have been presumed to be Bradyrhizobium (van Berkum et al., 2006). To date, on the chromosome, since no plasmids could be however, Bradyrhizobium strains capable of nodu- detected in the strains examined by using standard lating Aeschynomene have not been given specific plasmid isolation procedure (Baginsky et al., 2002; species status. Brito et al., 2005). A comprehensive description of the structural and In addition to symbiotic plasmids, several rhizo- functional genomics of strain BTAil was recently bial strains also contain one or more smaller, described, revealing the presence of a 229-kb accessory plasmids, also termed non-symbiotic accessory plasmid (Giraud et al., 2007). The pre- plasmids (Mercado-Blanco and Toro, 1996; Gonza- sence of plasmids in Bradyrhizobium sp. strains lez et al., 2006; Stiens et al., 2006; Young et al., capable of nodulating Aeschynomene appears not to 2006). Some of these plasmids have been shown to be a common feature of this group of bacteria, as no transfer between populations of indigenous rhizobia plasmid was identified in the closely related strain (Mercado-Blanco and Toro, 1996). The acquisition of ORS278 (Giraud et al., 2007). In this study we used some accessory plasmids has been correlated with in silico, physiological and in planta analyses to enhanced symbiotic performance (Martı´nez et al., understand the mode of regulation, phylogenetic 1987), while others have been shown to enhance affiliation and potential environmental role of the growth under