High-quality permanent draft genome sequence of the Bradyrhizobium elkanii type strain USDA 76T, isolated from Glycine max (L.) Merr
- Wayne Reeve1Email author,
- Peter van Berkum2,
- Julie Ardley1View ORCID ID profile,
- Rui Tian1,
- Margaret Gollagher3,
- Dora Marinova3,
- Patrick Elia2,
- T. B. K. Reddy4,
- Manoj Pillay5,
- Neha Varghese4,
- Rekha Seshadri4,
- Natalia Ivanova4,
- Tanja Woyke4,
- Mohamed N. Baeshen6,
- Nabih A. Baeshen7 and
- Nikos Kyrpides4, 7
© The Author(s). 2017
Received: 14 October 2016
Accepted: 21 February 2017
Published: 4 March 2017
Bradyrhizobium elkanii USDA 76T (INSCD = ARAG00000000), the type strain for Bradyrhizobium elkanii, is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from an effective nitrogen-fixing root nodule of Glycine max (L. Merr) grown in the USA. Because of its significance as a microsymbiont of this economically important legume, B. elkanii USDA 76T was selected as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria sequencing project. Here the symbiotic abilities of B. elkanii USDA 76T are described, together with its genome sequence information and annotation. The 9,484,767 bp high-quality draft genome is arranged in 2 scaffolds of 25 contigs, containing 9060 protein-coding genes and 91 RNA-only encoding genes. The B. elkanii USDA 76T genome contains a low GC content region with symbiotic nod and fix genes, indicating the presence of a symbiotic island integration. A comparison of five B. elkanii genomes that formed a clique revealed that 356 of the 9060 protein coding genes of USDA 76T were unique, including 22 genes of an intact resident prophage. A conserved set of 7556 genes were also identified for this species, including genes encoding a general secretion pathway as well as type II, III, IV and VI secretion system proteins. The type III secretion system has previously been characterized as a host determinant for Rj and/or rj soybean cultivars. Here we show that the USDA 76T genome contains genes encoding all the type III secretion system components, including a translocon complex protein NopX required for the introduction of effector proteins into host cells. While many bradyrhizobial strains are unable to nodulate the soybean cultivar Clark (rj1), USDA 76T was able to elicit nodules on Clark (rj1), although in reduced numbers, when plants were grown in Leonard jars containing sand or vermiculite. In these conditions, we postulate that the presence of NopX allows USDA 76T to introduce various effector molecules into this host to enable nodulation.
KeywordsRoot-nodule bacteria GEBA-RNB Nitrogen fixation Bradyrhizobium Soybean Type III secretion system
Soybean (Glycine max) (L.) Merr. is the dominant and the most important commercial legume crop species, yielding food oil and animal meal as well as nutritious vegetable protein [1–3]. The plant was first introduced into USA agriculture during the mid-18th century and was mainly used as a forage crop until the 1920s . The development of new cultivars, along with technological advances in soybean processing and increased demand for soybean products, has led to major increases in production during the 20th century .
As with most papilionoid legumes, soybean engages in a symbiotic relationship with dinitrogen-fixing soil bacteria known as rhizobia and is able to obtain on average 50–60% of its required nitrogen through symbiotic nitrogen fixation . A greater understanding of the symbiosis between soybean and its cognate rhizobia is of direct relevance for maintaining environmentally sustainable high crop yields, which significantly contributes to the Sustainable Development Goals adopted in September 2015 as part of the UN’s development agenda ‘Transforming our world: the 2030 Agenda for Sustainable Development’ .
The soybean-nodulating bacteria, known as Rhizobium japonicum according to a 1929 classification scheme , were reclassified as Bradyrhizobium japonicum in 1982 because of several fundamental morphological and physiological differences with the genus Rhizobium . The bacteria isolated from nodules of soybean had previously been shown to be phenotypically diverse, even though they were grouped together in the species Bradyrhizobium japonicum. One of the major methods that demonstrated this diversity was serology, which was used to classify individual isolates into 17 distinct serogroups . This was accomplished by generating antisera to specific strains in the USDA collection in Beltsville and then using the sera to generate a serological scheme. One of the strains used to generate antisera was USDA 76T and all isolates that cross-reacted with the antiserum generated with this serotype strain were combined together in the 76 serogroup. The strain USDA 76T deposited in the Beltsville collection was a re-isolate from a greenhouse-grown plant inoculated with USDA 74 in Maryland. In turn, USDA 74 was a re-isolate of USDA 8 from a plant passage field test in California in 1956. The original parent culture of USDA 76T is USDA 8, which was isolated from soybean grown at the Arlington Farm, Virginia in 1915.
Differences among the soybean root nodule bacteria classified as B. japonicum were also demonstrated using molecular methods. Hollis et al.  reported the presence of three DNA homology groupings by analysis of 28 strains within the soybean rhizobia. Using this approach, nine of the 17 serogroups were assigned to three DNA homology groupings: group I, the closely related group Ia and the more divergent group II. Supporting evidence for these three groupings was obtained by Kuykendall et al. . By sequence analysis of the 16S rRNA genes, each of the 17 serotype strains representing the serogroups were also placed into three closely related groups  that matched their separation by DNA homology. Since soybean strains could be distinguished phenotypically and by several approaches in molecular biology, Kuykendall et al.  proposed that DNA homology group II strains be separated from B. japonicum as the species Bradyrhizobium elkanii , with USDA 76T as the type strain.
Because of these distinguishing characteristics and its significance as a microsymbiont of the economically important legume soybean, B. elkanii USDA 76T was selected as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria sequencing project [14, 15]. Here we present a summary classification and a set of general features for B. elkanii strain USDA 76T , together with a description of its genome sequence and annotation.
Classification and features
pH range; Optimum
Soil, root nodule of Glycine max (L. Merr)
0 to <2% (w/v) NaCl
Free living, symbiotic
Alexandria, Virginia, USA
Sample collection date
An investigation of the symbiotic properties of soybean began with the work of Brooks  in the late 19th century, when he observed that soybean grown in the fields of his experiment station in Massachusetts only nodulated when supplied with dust he had brought with him from Japan. This led to the theory that soybean-nodulating bacteria in the soils of the USA were imported from the Far East. Cotrell et al.  and Hopkins  reported the supporting evidence that soybean in Kansas nodulated with soil taken from the Massachusetts Experiment station, or in Illinois from soil collected from fields with a history of soybean cultivation. However, several decades later it became evident that rhizobia that nodulated native American legumes within the genera Apios , Amphicarpa , Crotalaria , Desmodium , Lespedeza , Baptisia , Cassia , Genista and Wisteria also nodulated soybean [26–28]. With the exception of USDA 6 and USDA 38, which are from Japan, all the remaining soybean serotype strains were recovered from nodules of soybeans grown in the USA, including USDA 8 (the original parent of USDA 76T ). Consequently, it is unclear whether these rhizobia obtained from nodules of USA-grown soybean originate from the Far East or are in fact native to the soils of America. Therefore, the possibility exists that USDA 76T may be able to nodulate and form a symbiosis with a wide variety of legumes, but this has not been thoroughly investigated. Unfortunately, the communication that included the proposal of USDA 76T as the type strain for B. elkanii did not include results of plant tests to describe its symbiotic range, but instead relied on distinction by phenotype and genotype . An indication of the possible American origin of USDA 76T is its reported effectiveness in symbiosis with the native Apios americana Medik. and use as an inoculum for this potential leguminous crop . Further evidence for this theory is the ability of USDA 76T to nodulate and fix nitrogen with the native American Amphicarpaea bracteata (L.) Fernald . USDA 76T effectively nodulates the promiscuous Vigna unguiculata (L.) Walp. (cowpea), but is unable to nodulate the tropical American legume Phaseolus lunatus L. (Lima bean), which forms nodules with various other strains of bradyrhizobia . To our knowledge, the only other reported information is that USDA 74 (parent of USDA 76T ) forms an effective symbiosis with Macroptilium atropurpureum (DC.) Urb. (Siratro) and Vigna unguiculata (L.) Walp .
In soybean, the Rj(s) or rj(s) genetic loci have been identified as controlling the ability of compatible rhizobia to nodulate with a particular cultivar (reviewed by Hayashi et al. ). USDA 76T is reported to form nodules (albeit in reduced numbers) on the cultivar Clark (rj1) and to nodulate and fix N2 with the isogenic lines BARC-2 and BARC-3, harboring the Rj4 and rj4 alleles, respectively, when tested in Leonard jars with sterile vermiculite or sand . The symbiotic characteristics of B. elkanii USDA 76T on a range of selected hosts are summarized in Additional file 2: Table S2.
Genome sequencing information
Genome project history
Genome sequencing project information of Bradyrhizobium elkanii strain USDA 76T
High-quality permanent draft
2× Illumina libraries; Std short PE & CLIP long PE
Illumina HiSeq2000, PacBio
Velvet version 1.1.05; Allpaths-LG version r38445; phrap, version 4.24
Gene calling methods
Prodigal 1.4; GenePRIMP
GenBank Date of Release
Apr 22, 2013
Source Material Identifier
USDA 76, USDA 8, USDA 74, ATCC 49852, DSM 11554, IFO (now NBRC) 14791, LMG 6134
Symbiotic N2 fixation, agriculture
Growth conditions and genomic DNA preparation
After recovery from permanent storage, the B. elkanii USDA 76T was streaked onto MAG solid medium and grown at 28 °C for 6 days to obtain well grown, well separated colonies, then a single colony was selected and used to inoculate 5 ml MAG broth. The culture was grown on a gyratory shaker (200 rpm) at 28 °C for 6 days. Subsequently 1 ml was used to inoculate 50 ml MAG broth and grown on a gyratory shaker (200 rpm) at 28 °C until an OD600nm of 0.6 was reached. DNA was isolated from the cells according to van Berkum . Final concentration of the DNA was set to 0.5 mg ml−1. Culture identity was confirmed by partial sequence analysis of several housekeeping genes and the 16S rRNA gene using the prepared DNA as template for PCR.
Genome sequencing and assembly
The draft genome of B. elkanii USDA 76T was generated at the DOE Joint genome Institute (JGI) using the Illumina technology . An Illumina short-insert paired-end library was constructed with an average insert size of 200 bp that when sequenced generated 312,796,730 reads. An Illumina long-insert paired-end library with an average insert size of 6505.78 +/− 3679.88 bp also was constructed that when sequenced generated 19,315,434 reads. The total amount of sequence data obtained with the Illumina was 34,177 Mbp. Library construction and sequence analysis were done at the JGI according to the protocols outlined on their website . The first of two initial drafts, assembled with Allpaths version r38445 , contained 81 contigs in 17 scaffolds and subsequently a consensus was computationally shredded into 10 Kbp overlapping fake reads (shreds). The second draft assembled with Velvet, version 1.1.05 , resulted in consensus sequences that were computationally shredded into 1.5 Kbp overlapping fake reads (shreds). The data were assembled again with Velvet using the shreds from the first Velvet assembly to guide the next assembly. The consensus from this second Velvet assembly was shredded into 1.5 Kbp overlapping fake reads. The fake reads from the Allpaths and both Velvet assemblies together with a subset of the Illumina CLIP paired-end reads were assembled using parallel Phrap, version 4.24 (High Performance Software, LLC). Potential errors in the assemblies were corrected by manual editing with Consed [41–43]. Gap closure was accomplished using repeat resolution software (Wei Gu, unpublished) and sequence analysis of bridging PCR fragments with PacBio technology (Cliff Han, unpublished). Gaps were closed and the quality of the final sequence was improved with 35 PCR PacBio consensus sequences. The total size of the genome is 9.5 Mbp and the final assembly is based on 34,177 Mbp of Illumina draft data, which provides an average 3560x coverage of the genome.
Genes were identified using Prodigal  that was followed by a round of manual curation using GenePRIMP  as part of the DOE-JGI genome annotation pipeline [46, 47]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) non-redundant, UniProt, TIGRFam, Pfam, KEGG, COG, and InterPro databases. The tRNAScanSE tool  was used to find tRNA genes. Ribosomal RNA genes were found by searches against models of the ribosomal RNA genes built from SILVA . Other non–coding RNAs such as the RNA components of the protein secretion complex and the RNase P were identified by searching the genome for the corresponding Rfam profiles using INFERNAL . Additional gene prediction analysis and manual functional annotation were done within the Integrated Microbial Genomes-Expert Review system  developed by the Joint Genome Institute, Walnut Creek, CA, USA.
Genome statistics for Bradyrhizobium elkanii USDA 76T
% of Total
Genome size (bp)
DNA coding (bp)
DNA G + C (bp)
Protein coding genes
Genes in internal clusters
Genes with function prediction
Genes assigned to COGs
Genes with Pfam domains
Genes with signal peptides
Genes with transmembrane helices
Number of protein coding genes of Bradyrhizobium elkanii USDA 76T associated with the general COG functional categories
Translation, ribosomal structure and biogenesis
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, cell division, chromosome partitioning
Signal transduction mechanisms
Cell wall/membrane/envelope biogenesis
Intracellular trafficking, secretion, and vesicular transport
Posttranslational modification, protein turnover, chaperones
Energy production and conversion
Carbohydrate transport and metabolism
Amino acid transport and metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolite biosynthesis, transport and catabolism
General function prediction only
Not in COGS
Insights from the genome sequence
Using the Phylogenetic Profiler tool, 7556 genes were found to be conserved in five B. elkanii strains (587, CCBAU43297, CCBAU05737, USDA 76T , USDA 94), including genes encoding a general secretion pathway and type II, III, IV and VI secretion system proteins. The Type III secretion system (T3SS)  can either promote or impair the establishment of symbiosis, depending on the legume host , and has been characterized as a host determinant for rj1, Rfg1, Rj2 and Rj4 soybean cultivars [33, 57, 58]. The dominant soybean genes Rj2 and Rj4 restrict nodulation with specific strains of Bradyrhizobium . Most investigations of soybean host genes controlling the symbiosis have focused on the Rj4 soybean line that was originally identified by its inability to nodulate with USDA 61 ( B. elkanii , serogroup 31) . The predicted Rj4 thaumatin-like protein is thought to be involved in conferring resistance to Bradyrhizobium strains producing specific T3SS effector proteins . However, USDA 76T was reported to nodulate and form an effective nitrogen-fixing symbiosis with the isogenic lines BARC-2 (Rj4) and BARC-3 (rj4) [30, 61], suggesting that this strain does not produce the interacting T3SS effector protein(s). Conversely, the recessive soybean gene rj1rj1 , encoding a putative truncated Nod factor receptor protein , restricts nodulation by many Bradyrhizobium and Ensifer strains, although specific strains of B. elkanii , including USDA 76T , can form a limited number of nodules when tested with plants in Leonard jars containing sterilized vermiculite or sand [30, 59, 61].
B. elkanii USDA 76T originated from strain USDA 8, which was obtained in 1915 from an effective nodule of soybean grown on the USDA Arlington farm in Virginia. Its ability to nodulate the native North American legumes Apios americana Medik. and Amphicarpaea bracteata (L.) Fernald indicates a possible North American origin for this strain. USDA 76T was selected for genome sequencing  because of its significance as a microsymbiont of soybean. The genome size of USDA 76T was established as 9.5 Mbp, which falls within the range of 7.7 to 10.5 Mbp observed for other bradyrhizobial genomes. The genome of this N2-fixing microsymbiont contains nod, nif and fix genes located on an integrated symbiotic island, and genes encoding both an intact and an incomplete phage. According to ANI values, strain USDA 76T formed an ANI clique with four other B. elkanii soybean strains: USDA 94, 587, CCBAU 43297 and CCBAU 05737. Of particular interest was the discovery that these strains contain a T3SS that contains the NopCA pilus genes and the NopX translocon protein, which are essential for introducing effector molecules into host cells . The T3SS has been shown to be an important host range determinant that enables the nodulation of some soybean cultivars and is detrimental to symbiosis with other cultivars . Here we postulate that the presence of a functional T3SS is important in determining the host range of USDA 76T and enables it to form some nodules on the soybean cultivar Clark (rj1) when grown in Leonard jars with sterilized vermiculite or sand [65, 66]. Further analyses of Bradyrhizobium genomes, including that of USDA 76T , will increase our understanding of determinants that lead to the establishment and functioning of different Bradyrhizobium symbioses.
- ½ LA:
½ Lupin Agar
Average Nucleotide Identity
Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria
Integrated Microbial Genomes
Modified Arabinose Gluconate
Type 3 Secretion System
We thank Gordon Thompson (Murdoch University) for the preparation of SEM and TEM photos.
This work was performed under the auspices of the US Department of Energy’s Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231. We gratefully acknowledge the funding received from the Curtin University Sustainability Policy Institute, and the funding received from Murdoch University Small Research Grants Scheme in 2016.
PVB supplied the strain, background information for this project and the DNA to the JGI; TR performed all imaging; PVB, TR, JA and WR drafted the paper; MNB and NAB provided financial support and MG, DM, PE, TBKR, VM, NI, TW, RS and NK were involved in sequencing the genome and/or editing the final paper. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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