High-quality permanent draft genome sequence of Ensifer sp. PC2, isolated from a nitrogen-fixing root nodule of the legume tree (Khejri) native to the Thar Desert of India

The genus Prosopis (family Leguminosae , sub-family Mimosoideae [1]) comprises about 44 species that are widely distributed in the world’s semi-arid regions, mostly in North and South America with a few species found in Africa and south west Asia [2–4]. Several species have been widely introduced throughout the world over the last 200 years [5]. Prosopis may have evolved from P. africana (Guill. & Perr.) Taub., in which various character traits and small genome size (392–490 Mbp) indicate that it is a primitive species [2]. According to Burkart [2], Prosopis is an old genus that diverged early into several principal lineages, with some of these lineages producing more recent episodes of speciation. This is supported by a recent molecular dating analysis that places the divergence of the New World Prosopis Sections during the Oligocene (33.9 to 23.03 Mya) [6], which is remarkably ancient considering that the subfamily Mimosoideae originated between 42–50 Mya [7]. Section Prosopis consists of three species, Prosopis cineraria (L.) Druce, P. farcta (Banks et Sol.) Eig. and P. koelziana Burkart, which are native to North Africa and Asia [6].

P. cineraria is endemic to arid and semi-arid regions of the Indian Thar Desert and is designated as the state tree of Rajasthan [8]. It symbolizes the sacred mythological “Kalpa Vriksh” (wish tree) of the desert and is historically important, as it has been worshiped since ancient times by many rural communities in these arid regions. P. cineraria is a multipurpose tree used as food, fodder, shelter and medicine by the local inhabitants. It is an important component of agro forestry, agrisilvicultural and silvopastoral systems in the alkaline soil of the Thar Desert. The tree is extremely drought and salt tolerant, having a deep root system (>100 metres) that helps in acquiring nutrients and moisture from deeper soil layers. It produces green pods that are rich in nutrients and antioxidants and eaten as a vegetable in the hot summer [9]. P. cineraria is a good candidate for rehabilitation of dry, marginal or degraded lands of low fertility and/or high salinity. It plays a vital role as a soil binder in the stabilization of sand dunes and enriches poor desert soil by fixing atmospheric nitrogen in association with its rhizobial microsymbionts [10–13].

Prosopis is a promiscuous genus, being nodulated by a wide range of taxonomically diverse rhizobia. Mesquite (Torr.) in the Sonoran Desert, California is nodulated by diverse strains of fast- and slow-growing rhizobia [14]. Mesorhizobium chacoense CECT 5336^T is a microsymbiont of Prosopis alba Griseb. growing in the Chaco Arido region in Argentina [15], whereas in Spain is nodulated by strains of Ensifer medicae , E. meliloti and Rhizobium giardinii [16]. In Africa, the introduced Prosopis species P. chilensis (Molina) Stuntz, P. cineraria, P. juliflora (Sw.) DC. and P. pallida (Willd.) Kunth are reported to nodulate with strains of Ensifer arboris , E. kostiense, E. saheli and E. terangae [17, 18] and P. juliflora also forms effective symbioses with strains of Mesorhizobium plurifarium [19] and Rhizobium etli [20]. Nodulation of P. cineraria growing in its native range was first described by Basak and Goyal [10]. Recently, P. cineraria and other native legumes growing in the alkaline soils of the Thar desert have been reported to nodulate with a dominant novel group of Ensifer strains (PC2, TW10, TP13, RA9, TV3 and TF7) that are closely related to African and Australian Ensifer strains on the basis of 16S rRNA sequence similarity, but form a distinct, well-separated cluster [21, 22].

The indigenous rhizobia of wild tree legumes growing in such arid and harsh environments have superior tolerance to abiotic factors such as salt stress, elevated temperatures and drought and can be used as inoculants for wild as well as crop legumes cultivated in reclaimed desert lands [10]. Because of its ability to nodulate the keystone species P. cineraria as well as crop legumes such as Vigna radiata (L.) R.Wilczek and V. unguiculata (L.) Walp. [21], strain PC2 has therefore been selected as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) sequencing project [23]. Here we present a summary classification and a set of general features for Ensifer sp. strain PC2, together with a description of its genome sequence and annotation.

With a genome totaling 8.5 Mbp in size, Ensifer sp. PC2 is approximately 25 % larger than the average Ensifer genome in GenBank. Although PC2 shares 100 % 16S rRNA sequence identity and 99.17 Average Nucleotide Identity with Ensifer sp. TW10, also isolated from a Thar Desert woody legume, the genome of TW10 has a smaller size of 6.8 Mbp. PC2 contains over 1,000 genes that are not found in TW10, including two plasmid replication initiator proteins and a suite of genes (vir/trb) involved in conjugative transfer. From this it is assumed that the PC2 genome is multipartite and contains at least one conjugative plasmid. In PC2, 38.64 % of genes have not been assigned to a COG functional category, whereas in TW10, only 31.55 % have not been assigned to a COG functional category. Compared with TW10, PC2 has a much higher number of genes assigned to the mobilome category (54 and 120 genes, respectively) and to extracellular structures (29 and 44 genes, respectively).

Based on the 16S rRNA gene alignment, Ensifer sp. PC2 is most closely related to Ensifer sp. TW10 and Ensifer sp. WSM1721, two strains isolated from perennial legumes growing in arid climates and alkaline soils in India and Australia, respectively [21, 42]. Ensifer fredii strains isolated from Chinese soybean were also superdominant in sampling sites with alkaline-saline soils [43], which suggests that the biogeographic distribution of several Ensifer spp. is linked to their adaptation to alkaline soils. Further, this suggests that the symbiotic associations formed by promiscuous legumes, such as Prosopis , are likely to vary depending on which rhizobial genera are best adapted to the edaphic conditions in which the host is growing.

The ability of PC2 to fix nitrogen with both P. cineraria (L.) Druce and the crop legumes Vigna radiata (L.) R.Wilczek and V. unguiculata (L.) Walp. makes it a valuable inoculant strain for use in arid, alkaline regions such as the Thar desert. Analysis of the PC2 sequenced genome and comparison with the genomes of sequenced Ensifer spp. and other rhizobia will provide insights into the molecular basis of the patterns seen in rhizobial biogeographic distributions and associations with plant hosts and into the molecular determinants of rhizobial tolerance to arid and alkaline environments.